ES2832546T3 - Immunogenic WT-1 Peptides and Methods of Using Them - Google Patents

Abstract

An isolated peptide consisting of the amino acid sequence NLMNLGATL (SEQ ID NO: 6).

Description

DESCRIPTION

Immunogenic WT-1 Peptides and Methods of Using Them

Field of the invention

This invention provides peptides, compositions, and vaccines comprising the same, and their use to treat, reduce the incidence, and induce immune responses to a WT1-expressing cancer.

Background of the invention

Wilms tumor (WT), a pediatric nephroblastoma that occurs with a frequency of 1 in 10,000 births, has been the subject of intensive basic and clinical research for several years. The tumor is of embryonic origin, it is detected in children usually during the first 5 years of life, and it can present unilaterally or bilaterally. A WT arises when the condensed metanephric mesenchymal cells of the developing kidney fail to differentiate properly. The implication of the Wilms tumor suppressor gene 1 (WT1) in the etiology of WT illustrated the impact that genetic alterations can have on both development and tumorigenesis.

Wilms tumor protein I (WT1) is a zinc finger transcription factor that is expressed during normal ontogenesis, such as in the kidney, testes, and fetal ovaries. In adults, WTI expression is limited to low levels in hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes, and some cells in the testes and ovaries. The recent demonstration that WTI is overexpressed in various types of leukemia suggested that WTI would be an attractive target for immunotherapy for various cancers.

Wilms tumor oncogenic protein (WT1) is an attractive target for immunotherapy for leukemias and a wide range of cancers. Peptides derived from the WT1 protein have been identified that induce cytotoxic CD8 T cells restricted by HLA-A0201, capable of killing tumor cells. Two peptides that bind to HLA-A0201 (RMFPNAPYL; SEQ ID NO: 56) or HLA-A2402 (CMTWNQMNL; SEQ ID NO: 57) have been extensively studied worldwide and clinical trials have been conducted in patients with leukemia and other solid tumors (Oka et al. Scientific World Journal 2007; 7: 649-665; Mundlos et al. Development 1993; 119: 1329-41; Keilholz et al. Leukemia 2005; 19: 1318-1323). These results are encouraging and have provided strong evidence and rationale for therapeutic targeting of WT1-derived T-cell epitopes for leukemias and a wide range of human cancers.

The therapeutic application of the above two WT1-derived peptides is limited to individuals who are HLA-A0201, an HLA haplotype found in approximately 40% of Caucasians, and HLA-A2402, a haplotype found in approximately 40% of Caucasians. 40% of the Japanese and other Asian populations. Therefore, there is an unmet need for WT1 derived peptides that could be used for most of the world's populations. To extend therapeutic application to a broader range of the population, new peptides derived from the WT1 protein that bind to multiple HLA haplotypes are desired. Said peptides would therefore be capable of stimulating the T cells of a greater percentage of the target population, which would allow a vaccine strategy that would target a large segment of the population, with durable cytotoxic memory cells.

WO 2007/120673 describes immunogenic WT-1 peptides, immunogenic compositions and vaccines comprising the peptides, and methods for treating, reducing the incidence and inducing immune responses to a WT1 expressing cancer, using the peptides and compositions. .

Brief description of the invention

This application describes immunogenic peptides, compositions, and compositions such as vaccines comprising immunogenic peptides and methods for treating, reducing the incidence, and inducing immune responses to a WT1-expressing cancer, comprising administering immunogenic peptides or stimulating T cells outside of a human patient. which can then be infused into the patient for treatment.

In one embodiment, the present invention provides an isolated peptide consisting of an amino acid sequence (AA) selected from SEQ ID NO: 6, 14, 15, 16, 17, and 22. In one embodiment, the isolated peptide binds to a HLA class I molecule and / or an HLA class II molecule. In one embodiment, the present invention provides an isolated HLA class I binding peptide having the sequence of SEQ ID NO: 6. In one embodiment, the present invention provides an isolated HLA class II binding peptide WT1 comprising an amino acid sequence (AA) selected from SEQ ID NO: 14, 15, 16 and 17. These peptides can be referred to as peptides of the invention.

The present invention also provides a nucleic acid molecule encoding a peptide of the invention. The present invention also provides a vector, such as an attenuated virus vector or a Salmonella vector. typhi, which comprises the nucleic acid molecule. The vector may further encode any of interleukins 1 to 15, interferon alpha, beta or gamma, tumor necrosis factor, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), factor granulocyte colony stimulating (G-CSF), neutrophil activating protein (NAP), macrophage activating and chemotactic factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia or MIP-Ib, a complement component, or combinations thereof.

The present invention also provides a pharmaceutical composition comprising a peptide of the invention, or a nucleic acid molecule encoding the peptide, or a vector comprising the nucleic acid molecule and a pharmaceutically acceptable carrier, vehicle or excipient.

In another embodiment, the present invention provides a vaccine comprising one or more peptides of the invention, and optionally, an antigen presenting cell (APC) and / or an adjuvant, diluent or carrier and / or at least one peptide derived from WT1. additional. In one embodiment, the adjuvant is QS21, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, alum, a growth factor, a cytokine, a chemokine, an interleukin, Montanide ISA 51, or GM-CSF.

In another embodiment, the present invention provides a pharmaceutical composition or vaccine of the invention for use in treating a subject with a WT1-expressing cancer or reducing the incidence of a WT1-expressing cancer, or its relapse.

In another embodiment, the present invention provides a composition comprising (a) an antigen presenting cell (APC) and (b) a peptide of the invention, or a nucleic acid molecule encoding the peptide, or a vector comprising the nucleic acid molecule.

In another embodiment, the present invention provides the use of a peptide or vaccine or composition of the invention, or a nucleic acid molecule encoding the peptide, or a vector comprising the nucleic acid molecule, for in inducing I vitro formation and proliferation of CTL specific for cancer cells expressing WT1.

In one embodiment, the WT1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, a cancer of the ovary, uterine cancer, thyroid cancer, liver cancer, kidney cancer, Kaposi's sarcoma, sarcoma, hepatocellular carcinoma, Wilms tumor, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS) ) or non-small cell lung cancer (NSCLC). In one embodiment, the WT1-expressing cancer is mesothelioma.

Brief description of the figures

In the figures of the present disclosure, the set of data bars grouped in the graphs for each peptide is presented in the same order from left to right as shown in the figure legend from top to bottom.

Figure 1 shows the results of a T2 stabilization assay showing that the binding of the NLMNLGATL peptide to the HLA-A2 molecule is stronger than that of the n Qm peptides NLGATL and NYMNLGATL. Native NQMNLGATL, heteroclite NLMNLGATL on Ym NLGATL was pulsed on T2 cells at the indicated concentrations as described in Materials and Methods. The stabilization of the HLA-A2 molecule by the peptides was measured by the expression of the HLA-A2 molecule;

Figure 2 AB shows that the NLMNLGATL peptide induces a strong peptide-specific T cell response that cross-reacts with its native sequence NQMNLGATL. For each peptide, the bars represent, from left to right, CD14, native peptide, NLMNLGATL, and PSMA, respectively. CD3 T cells from a healthy HLA-A0201 homozygous donor were stimulated with either a NQM or NLMNLGATL peptide for 3 (A) or 5 (B) rounds. The specific response for the peptide was measured by IFN-g secretion after challenge with the individual peptide. Each data point represents the mean / - SD of the triplicate cultures;

Figure 3 shows that the NLMNLGATL peptide induces T cell cytotoxicity against WT1 + HLA-A0201 + leukemia cells. T cells from an HLA-A0201 positive donor were stimulated with peptide NLMNLGATL for 5 rounds. The cytotoxicity of the cells was measured by a 51Cr-5 h release assay against the AML cell line SET-2 (WT1 +, HLA-A0201 +), HL-60 (WT1 +, HLA-A0201-) or primary leukemia blasts. of an HLA-A2 positive patient. Each data point represents the mean / - SD of the triplicate cultures;

Figure 4 AB shows the peptide-specific T cell response in donor HLA-A2402. CD3 T cells from a healthy HLA-A2402 homozygous donor were stimulated with NQMNLGATL, NLMNLGATL or NYMNLGATL peptides for 3 (A) or 5 (B) rounds. For each peptide, the bars represent, from left to right, CD14, native peptide, NLMNLGATL, NYMNLGATL, and irrelevant peptide, respectively. The specific answer for the peptide was measured by IFN-g secretion after challenge with the individual peptide. Each data point represents the mean / - SD of the triplicate cultures;

Figure 5 AB shows specific T cell responses for the HLA-DR.B1 peptide. (TO). CD3 T cells were stimulated with either DR-het-1 or DR-het-2 peptide for 5 rounds and the specific response for the epitope was measured by the IFN-g Elispot assay. For each peptide, the bars represent, from left to right, CD14, native peptide, NLMNLGATL, WT1-CMT, PSMA, DR-Nat-1 / Nat-2, DR-het-1 / het-2, B2A2L, BA25 and HL-60, respectively. (B). CD3 T cells were stimulated with short peptides NQMNLGATL, NLMNLGATL and long peptides DR-native-1, DR-native-2, DR-het-1 or DR-het-2 for 5 rounds and cytotoxicity was measured by the assay of 51Cr release, against BA-25 leukemia cell line (HLA-A2 + / A24 +, WT1 +) and HL-60 control cells. Each data point represents the mean / - SD of the triplicate cultures;

Figure 6 depicts CD3 T cells from an HLA-B0702 positive donor that were stimulated with 2 sets of peptides (five in total) for 5 times in vitro. The specific response for the peptide was measured by the IFN-gamma ELISPOT assay, against a single peptide; peptides tested from left to right are SEQ ID NOS: 34, 37, 38, 30 and 31; and for each peptide, the bars represent, from left to right, the responses to CD14, native peptide-1, Het-1 to native-1, Het-2 to native-1, native-2, Het-1 to native- 2 and control, respectively; Y

Figure 7 represents the results of an ELISPOT assay using donor SA (5 stimulations) for the A24 peptide Het-1 (SEQ ID NO: 6) and Het-2 (SEQ ID NO: 7), compared to the native sequence (SEQ ID NO: 5). For each peptide, the bars represent, from left to right, CD14, native peptide-A24, A24-het-1, A24-het-2, A24-235, and PSMA, respectively. Heterocyte peptides generate cross-reactive responses.

Detailed description of the invention

This application describes immunogenic peptides, and compositions and vaccines comprising immunogenic peptides, and methods for treating, reducing the incidence, and inducing immune responses to a WT1-expressing cancer, comprising administering one or more immunogenic peptides.

This application describes synthetic peptides and methods for treating, reducing the incidence, and inducing immune responses against a WT1-expressing cancer, comprising immunogenic peptides.

The WT1 molecule from which the peptides of the present invention are derived has, in another embodiment, the sequence:

1 SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA AQAPGPRRLL

51 AAILDFLLLQ DPASTCVPEP ASQHTLRSGP GCLQQPEQQG VRDPGGIWAK

101 LGAAEASAER LQGRRSRGAS GSEPQQMGSD VRDLNALLPA VPSLGGGGGC

151 ALPVSGAAQW APVLDFAPPG ASAYGSLGGP APPPAPPPPP PPPPHSFIKQ

201 EPSWGGAEPH EEQCLSAFTV HFSGQFTGTA GACRYGPFGP PPPSQASSGQ

251 ARMFPNAPYL PSCLESQPAI RNQGYSTVTF DGTPSYGHTP SHHAAQFPNH

301 SFKHEDPMGQ QGSLGEQQYS VPPPVYGCHT PTDSCTGSQA LLLRTPYSSD

351 NLYQMTSQLE CMTWNQMNLG ATLKGVAAGS SSSVKWTEGQ SNHSTGYESD

401 NHTTPILCGA QYR1HTHGVF RGIQDVRRVP GVAPTLVRSA SETSEKRPFM

451 CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF SRSDQLKRHQ

501 RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGKTSEKPF SCRWPSCQKK

551 FARSDELVRH HNMHQRNMTK LQLAL (SEQ I> NO: 51).

The previous sequence of the WT-1 protein is that published by Gessler et al., (Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH, Bruns GA. Homozygous deletion in Wilms tumors of a zinc-finger gene identified by chromosome jumping. Nature. 1990; 343 (6260): 774-778. Previously published 02/22/1990 as DOI 10.1038 / 343774a0.) which comprises 575 amino acids and includes the first 126 amino acids at the N-terminus that are missing in the isoform (Exon 5+, KTS +) of WT-116.

In another embodiment, the sequence of WT1 is

MGSDVRD1NALLPA VPSLGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPP

PPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQA

RMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGS

LGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLK

GVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYR1HTHGVFRGIQDVRRVPGVAPTL

VRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLK

RHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVR

HHNMHQRNMTKLQLAL (GenBanK Accession No. AY245105; SEQ ID NO: 52).

In another embodiment, the WT1 molecule has the sequence:

AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAP

VLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQF

TGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHT

PSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDN

LYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYR1HTHGVFRGIQDVRRV

PGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRF

SRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARS

DELVRHHNMHQRNMTKLQLAL (GenBank Accession No. NM_000378; SEQ ID NO: 53).

In another embodiment, the WT1 molecule has the sequence:

MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGA

SGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGP

APPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQ

ASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDP

MGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQM

NLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYR1HTHGVFRGIQDVRRV

PGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRF

SRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARS

DELVRHHNMHQRNMTKLQLAL (GenBank Accession No. NP_077742; SEQ ID NO: 54).

In another embodiment, the WT1 protein has the sequence set forth in GenBank, no. accession NM_024426. In other embodiments, the WT1 protein has or comprises one of the sequences set in one of the following sequence entries: NM_024425, NM_024424, NM_000378, S95530, D13624, D12496, D 12497, or X77549. In another embodiment, the WT1 protein has any other WT1 sequence known in the art. This application describes immunogenic peptides, compositions and compositions such as vaccines comprising immunogenic peptides and methods for treating, reducing the incidence and inducing immune responses to a WT1 expressing cancer, comprising the administration of immunogenic peptides. In some cases, the peptides described in the present description are derived from peptides that are native sequences of WT1, and may be referred to herein as WT1 derived peptides or as a WT1 peptide.

In one embodiment, the present invention provides an isolated WT1 peptide consisting of the amino acid sequence (AA) NLMNLGATL (SEQ ID NO: 6). In another embodiment, the present invention provides an isolated WT1 peptide consisting of an amino acid sequence selected from CMTWNLMNLGATLKG (SEQ ID NO: 14), MTWNLMNLGATLKGV (SEQ ID NO: 15), TWNLMNLGATLKGVA (SEQ ID NO: 16), WNLMNLG ( SEQ ID NO: 17) and CMTWNLMNLGATLKGVA (SEQ ID NO: 22). In one embodiment, the isolated peptide binds to an HLA class I molecule, an HLA class II molecule, or a combination thereof. In one embodiment, the present invention provides an isolated HLA class I-binding peptide WT1 having the amino acid sequence NLMNLGATL (SEQ ID NO: 6). In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid sequence selected from CMTWNLMNLGATLKG (SEQ ID NO: 14), MTWNLMNLGATLKGV (SEQ ID NO: 15), TWNLMNLGATLKGVA (SEQ ID NO : 16) and WNLMNLGATLKGVAA (SEQ ID NO: 17). These peptides can be referred to as peptides of the invention.

In another embodiment, the present invention provides a composition comprising (a) an antigen presenting cell and (b) a peptide of the invention.

In another embodiment, the present invention provides a vaccine comprising one or more peptides of the invention.

In another embodiment, the present invention provides a WT1 peptide or vaccine of the present invention for use in treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a WT1 peptide or vaccine of the present invention for use in reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present invention provides an immunogenic composition comprising a WT1 peptide of the present invention or a nucleotide molecule encoding the WT1 peptide for use in inducing an immune response against cancer in a subject.

In another embodiment, the present invention provides an immunogenic composition comprising a WT1 peptide of the present invention or a nucleotide molecule encoding the WT1 peptide for use in treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides an immunogenic composition comprising a WT1 peptide of the present invention or a nucleotide molecule encoding the WT1 peptide for use in reducing the incidence or relapse of a WT1-expressing cancer, on a subject.

In one embodiment, the WT1-expressing cancer is acute myelogenous leukemia (AML). In another embodiment, WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematologic cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is gastric cancer. In another embodiment, the WT1-expressing cancer is colon cancer. In another embodiment, the WT1-expressing cancer is lung cancer. In another embodiment, the WT1-expressing cancer is breast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is ovarian cancer. In another embodiment, the WT1-expressing cancer is uterine cancer. In another embodiment, the WT1-expressing cancer is thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is thyroid cancer. In another embodiment, the WT1-expressing cancer is liver cancer. In another embodiment, the WT1-expressing cancer is kidney cancer. In another embodiment, the WT1-expressing cancer is Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.

In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with lung cancer. In another embodiment, the solid tumor is associated with breast cancer. In another embodiment, the solid tumor is associated with colorectal cancer. In another embodiment, the solid tumor is associated with prostate cancer. In another embodiment, the solid tumor is associated with ovarian cancer. In another embodiment, the solid tumor is associated with kidney cancer. In another embodiment, the solid tumor is associated with pancreatic cancer. In another embodiment, the solid tumor is associated with brain cancer. In another embodiment, the solid tumor is associated with gastrointestinal cancer. In another embodiment, the solid tumor is associated with skin cancer. In another embodiment, the solid tumor is associated with a melanoma.

In another embodiment, the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT1-derived peptide. In certain embodiments, a composition comprising at least 2 different peptides is provided. In certain embodiments, a composition comprising at least 3 or at least 4 different peptides is provided. In certain embodiments, the composition of the present invention is a vaccine.

In another embodiment, the present invention provides a peptide or composition of the present invention for use in treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a peptide or composition of the present invention for use in reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.

In another embodiment, the present application describes a method for inducing the formation and proliferation of CTL specific for the WT1 protein, the method comprises contacting a population of lymphocytes with a peptide or composition of the present invention, thereby inducing the formation and proliferation of a specific CTL of the WT1 protein. This method can be performed in vitro, ex vivo or in vivo. When performed in vitro or ex vivo, these CTLs can then be infused into a patient to obtain a therapeutic effect.

In another embodiment, the present application describes a method for inducing the formation and proliferation of (a) a CD8 + lymphocyte specific for the WT1 protein; or (b) a CD4 + lymphocyte specific for the WT1 protein, or the combination thereof, the method comprises contacting a population of lymphocytes with a peptide or composition of the present invention, thereby inducing the formation and proliferation of (a) a CD8 + lymphocyte specific for the WT1 protein; or (b) a CD4 + lymphocyte specific for the WT1 protein; or a combination thereof. This method can be performed in vitro, ex vivo or in vivo. When performed in vitro or ex vivo, these CTLs can then be infused into a patient to obtain a therapeutic effect.

Therefore, the present invention provides the use of a peptide or nucleic acid molecule or vector or composition of the present invention to induce the formation and proliferation of WT1-expressing cancer cell-specific CTLs in vitro.

"Peptide", in another embodiment of the methods and compositions of the present invention, refers to a compound of AA subunits connected by peptide bonds. In another embodiment, the peptide comprises an AA analog. In another embodiment, the peptide comprises a peptidomimetic. The different AA and peptidomimetic analogs that can be included in the peptides of the methods and compositions of the present invention are listed below. In another embodiment, the subunits are joined by peptide bonds. In another embodiment, the subunit is attached by another type of bond, eg, ester, ether, etc.

The unaltered peptides of the present invention (as described both above and below) are collectively referred to herein as "WT1 peptides". Each of the modalities listed below for "WT1 peptides" applies to unaltered WT1 peptides and HLA class I and class II heteroclite peptides of the present invention.

In another embodiment, a WT1 peptide of the present invention binds to an HLA class I molecule or a class II molecule. In another embodiment, the peptide binds to both a class I and a class II molecule. In another embodiment, the HLA class II molecule is an HLA-DRB molecule. In another embodiment, the HLA class II molecule is an HLA-DRA molecule. In another embodiment, the HLA molecule is an HLA-DQA1 molecule. In another embodiment, the HLA molecule is an HLA-DQB1 molecule. In another embodiment, the HLA molecule is an HLA-DPA1 molecule. In another embodiment, the HLA molecule is an HLA-DPB 1 molecule. In another embodiment, the HLA molecule is an HLA-DMA molecule. In another embodiment, the HLA molecule is an HLA-DMB molecule. In another embodiment, the HLA molecule is an HLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOB molecule. In another embodiment, the HLA molecule is any other HLA class II molecule known in the art.

In another embodiment, the HLA class I molecule whose binding motif is contained in or comprises a peptide of the present invention is, in another embodiment, an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-B molecule. In another embodiment, the HLA class I molecule is an HLA-C molecule. In another embodiment, the HLA class I molecule is an HLA-A0201 molecule. In another embodiment, the molecule is HLA A1. In another embodiment, the HLA class I molecule is HLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. In another embodiment, the HLA class I molecule is HLA A3. In another embodiment, the HLA class I molecule is HLA A3.2. In another embodiment, the HLA class I molecule is HLA A11. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8.

In another embodiment, the HLA class I molecule-binding WT1-derived peptide of the present invention binds to a superfamily of HLA class I molecules. In another embodiment, the superfamily is the A2 superfamily. In another embodiment, the superfamily is superfamily A3. In another embodiment, the superfamily is superfamily A24. In another embodiment, the superfamily is superfamily B7. In another embodiment, the superfamily is superfamily B27. In another embodiment, the superfamily is superfamily B44. In another embodiment, the superfamily is the C1 superfamily. In another embodiment, the superfamily is the C4 superfamily. In another embodiment, the superfamily is any other superfamily known in the art.

In another embodiment, the HLA molecule is a molecule A0101, A0201, A0203, A2402, A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001, B4402, B4701, B401701, C0B1701, C0B1701, C0B1701 , 0101, DRB-, 0402, DRB- | 0402, DRB- | 0401 or DRB- | 1104. In another embodiment, the peptides of the invention bind to the HLA class I or class II molecules described for each peptide in the Tables below. In one embodiment, certain peptides can bind to more than one HLA allele.

In another embodiment, a WT1 peptide of the methods and compositions of the present invention is designed to show affinity for an HLA molecule. In another embodiment, the affinity is a high affinity, as described in the present disclosure.

HLA molecules, otherwise known as major histocompatibility complex (MHC) molecules, bind to peptides and present them to immune cells. Thus, in another embodiment, the immunogenicity of a peptide is partially determined by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes.

In another embodiment, a peptide of the present invention is immunogenic. In another embodiment, "immunogenic" refers to the ability to stimulate, elicit, or participate in an immune response. In another embodiment, the immune response elicited is a cell-mediated immune response. In another embodiment, the immune response is a combination of humoral and cell-mediated responses.

In another embodiment, T cells that bind to the peptide-MHC molecule complex are activated and cells expressing a protein comprising the peptide are induced to proliferate and lyse. Typically, T cells are initially activated by "professional" antigen-presenting cells ("APCs"; eg, dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T-cell activation as opposed to anergy or apoptosis. In another embodiment, the response is heteroclite, as described herein, such that the CTL lyses a neoplastic cell that expresses a protein that has an AA sequence homologous to a peptide of this invention, or a different peptide than the one used. to stimulate the T cell first.

In another embodiment, an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and / or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, lead to a faster and more intense immune response. Said responses are measured, in another embodiment, by measuring the degree of proliferation of the population of T cells exposed to the peptide. In another embodiment, these responses are measured by any of the methods listed below.

In another embodiment, the peptides of the present invention bind to an HLA class II molecule with high affinity. In other embodiments, the HLA class II molecule is any HLA class II molecule listed in the present disclosure.

In another embodiment, the peptide derivatives of the present invention bind to an HLA class I molecule with high affinity. In other embodiments, the MHC class I molecule is any MHC class I molecule listed in the present disclosure.

In another embodiment, a peptide of the present invention binds to an HLA class II molecule with significant affinity, while a peptide derived from the parent peptide binds to an HLA class I molecule with significant affinity.

In another embodiment, "affinity" refers to the concentration of peptide necessary to inhibit the binding of a standard peptide to the indicated MHC molecule by 50%. In another embodiment, "high affinity" refers to an affinity that is such that a concentration of about 500 nanomolar (nM) or less of the peptide is required for a 50% inhibition of binding of a standard peptide. In another embodiment, a concentration of about 400 nM or less of the peptide is required. In another embodiment, the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM. In another embodiment, the binding affinity is 60 nM. In another embodiment, the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM. In another embodiment, the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM.

In another embodiment, "affinity" refers to a measure of the strength of binding to the MHC molecule. In another embodiment, the affinity is measured using a method known in the art to measure competitive binding affinities. In another embodiment, affinity is measured using a method known in the art to measure relative binding affinities. In another embodiment, the method is a competitive binding assay. In another embodiment, the method is a radioimmunoassay or RIA. In another embodiment, the method is a BiaCore analysis. In another mode, the method is any other method known in the art. In another embodiment, the method produces an IC50 relative to an IC50 of a reference peptide of known affinity.

In another embodiment, "high affinity" refers to an IC50 of 0.5 to 500 nM. In another embodiment, the IC50 is 1-300 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50 is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10-50 nM. In another embodiment, the IC50 is 20-50 nM. In another embodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30 nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment, the IC50 is 15-25 nM.

In another embodiment, a peptide of the present invention binds to a superfamily of HLA molecules. Superfamilies of HLA molecules share very similar or identical binding motifs. In another embodiment, the superfamily is an HLA class I superfamily. In another embodiment, the superfamily is an HLA class II superfamily.

The terms "HLA-binding peptide," "HLA class I molecule-binding peptide," and "HLA-class II molecule-binding peptide" refer, in another embodiment, to a peptide that binds to a HLA molecule with measurable affinity. In another embodiment, the terms refer to a peptide that binds to an HLA molecule with high affinity. In another embodiment, the terms refer to a peptide that binds to an HLA molecule with sufficient affinity to activate a T cell precursor. In another embodiment, the terms refer to a peptide that binds to an HLA molecule with sufficient affinity. to mediate recognition by a T cell. The HLA molecule is, in other embodiments, any of the HLA molecules listed in the present disclosure.

"Heteroclite" refers, in another embodiment, to a peptide that elicits an immune response that recognizes the original peptide from which the heteroclite peptide is derived (eg, the peptide does not contain the anchor residue or other residue mutations) . In another embodiment, "parent peptide" refers to a peptide of the present invention. In another embodiment, "heteroclite" refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclite peptide is derived, wherein the immune response generated by vaccination with the heteroclite peptide is greater than the response immune system generated by vaccination with the parent peptide. In another embodiment, a "heteroclite" immune response refers to an immune response that recognizes the original peptide from which the improved peptide is derived (eg, the peptide does not contain the anchor residue mutations). In another embodiment, a "heteroclite" immune response refers to an immune response that recognizes the original peptide from which the heteroclite peptide is derived, wherein the magnitude of the immune response generated by vaccination with the heteroclite peptide is greater than the immune response generated by vaccination with the parent peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclite peptide is greater than the immune response substantially equal to the response to vaccination with the parent peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclite peptide is greater than the immune response less than the response to vaccination with the parent peptide. In another embodiment, a heteroclite peptide of the present invention is a HLA class I heteroclite peptide. The methods for identifying HLA class I and class II residues, and for enhancing binding to HLA by mutation of the residues, are well known in the art, as described below.

In another embodiment, a heteroclite peptide of the present invention induces an immune response that increases at least 2-fold relative to the WT1 peptide from which the heteroclite peptide is derived ("native peptide"). In another embodiment, the increase is 3-fold over the native peptide. In another embodiment, the increase is 5-fold over the native peptide. In another embodiment, the increase is 7-fold over the native peptide. In another embodiment, the increase is 10-fold over the native peptide. In another embodiment, the increase is 15-fold over the native peptide. In another embodiment, the increase is 20-fold over the native peptide. In another embodiment, the increase is 30-fold over the native peptide. In another embodiment, the increase is 50-fold over the native peptide. In another embodiment, the increase is 100-fold over the native peptide. In another embodiment, the increase is 150-fold over the native peptide. In another embodiment, the increase is 200-fold over the native peptide. In another embodiment, the increase is 300-fold over the native peptide. In another embodiment, the increase is 500-fold over the native peptide. In another embodiment, the increase is 1000-fold over the native peptide. In another embodiment, the increase is more than 1000 fold over the native peptide.

In another embodiment, the present invention provides an HLA class II heteroclite peptide derived from an isolated WT1 peptide of the present invention. In another embodiment, the derivation process comprises introducing a mutation that enhances the binding of the peptide to an HLA class II molecule. In another embodiment, the derivation process consists of introducing a mutation that enhances the binding of the peptide to an HLA class I molecule. In another embodiment, the mutation is in an HLA class II anchoring residue. In another embodiment, a class II heteroclite peptide of the present invention is identified and tested in a manner analogous to the identification and testing of HLA class I heteroclite peptides, as exemplified in the present disclosure.

In another embodiment, the HLA class II binding site in a peptide of the present invention is created or enhanced by mutation of an anchor residue to the HLA class II motif. In another embodiment, the anchor residue that is modified is at position P1. In another embodiment, the anchor residue is at position P2. In another embodiment, the anchor residue is at position P6. In another embodiment, the anchor residue is at position P9. In another embodiment, the anchor residue is selected from positions P1, P2, P6, and P9. In another embodiment, the anchor residue is at position P3. In another embodiment, the anchor residue is at position P4. In another embodiment, the anchor residue is at position P5. In another embodiment, the anchor residue is at position P6. In another embodiment, the anchor residue is at position P8. In another embodiment, the anchor residue is at the P10 position. In another embodiment, the anchor residue is at position P11. In another embodiment, the anchor residue is at position P12. In another embodiment, the anchor residue is at position P13. In another embodiment, the anchor residue is at any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, p2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve binding to HLA class II.

In another embodiment, a heteroclite peptide is generated by introducing a mutation that creates an anchor motif. "Anchor motifs" or "anchor residues" refer, in another embodiment, to 1 or a set of preferred residues at particular positions in an HLA-binding sequence.

The HLA-binding sequence is a class II HLA-binding sequence. In another embodiment, the HLA-binding sequence is a class I HLA-binding sequence. In another embodiment, the positions corresponding to the anchor motifs are those that play a significant role in binding to the HLA molecule. In another embodiment, the anchoring residue is a primary anchoring motif. In another embodiment, the anchoring residue is a secondary anchoring motif.

Methods for predicting MHC class I and II epitopes are well known in the art. In one embodiment, the software from the Bioinformatics & Molecular Analysis Section (National Institutes of Health, Washington, DC) available at http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parker comboform is helpful. . This computer program classifies 9-mer or 10-mer peptides at a half-time dissociation coefficient predicted from HLA class I molecules (Pinilla, et al., Curr Opin Immunol, 11 (2): p. 193- 202 (1999)). In another embodiment, the MHC class II epitope is predicted using TEPITOPE (Meister GE, Roberts Cg et al., Vaccine 1995 13: 581-91). In another embodiment, the MHC class II epitope is predicted using the EpiMatrix (De Groot AS, Jesdale BM et al. AIDS Res Hum Retroviruses 199713: 529-31). In another embodiment, the MHC class II epitope is predicted using the Predict method (Yu K, Petrovsky N et al., Mol Med. 2002 8: 137-48). In another embodiment, the MHC class II epitope is predicted using the SYFPEITHI epitope prediction algorithm (Examples). In another embodiment, the MHC class II epitope is predicted using Rankpep. In another embodiment, the MHC class II epitope is predicted using any other method known in the art.

In another embodiment, in the case of HLA class II binding peptides (eg, HLA-DR binding peptides), the anchor residue that is modified is at the P1 position. In another embodiment, the anchor residue is at position P2. In another embodiment, the anchor residue is at position P6. In another embodiment, the anchor residue is at position P9. In other embodiments, the anchor residue is at the P3, P4, P5, P6, P8, P10, P11, P12, or P13 position. In another embodiment, the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve binding to HLA class II. In another embodiment, any combination of the above residues is mutated.

In another embodiment, a WT1 peptide of the present invention binds 2 different HLA class II molecules. In another embodiment, the peptide binds to three different HLA class II molecules. In another embodiment, the peptide binds to four different HLA class II molecules. In another embodiment, the peptide binds to five different HLA class II molecules. In another embodiment, the peptide binds to six different HLA class II molecules. In another embodiment, the peptide binds to more than six different HLA class II molecules.

In another embodiment, HLA class II molecules that are bound by a WT1 peptide of the present invention are encoded by two or more different alleles at a given HLA class II locus. In another embodiment, HLA class II molecules are encoded by 3 different alleles at one locus. In another embodiment, HLA class II molecules are encoded by 4 different alleles at one locus. In another embodiment, HLA class II molecules are encoded by 5 different alleles at one locus. In another embodiment, HLA class II molecules are encoded by 6 different alleles at one locus. In another embodiment, HLA class II molecules are encoded by more than six different alleles at a locus.

In another embodiment, HLA class II molecules linked by the WT1 peptide are encoded by HLA class II genes at 2 separate loci. In another embodiment, the linked HLA molecules are encoded by HLA class II genes at 2 or more other loci. In another embodiment, the linked HLA molecules are encoded by HLA class II genes at 3 separate loci. In another embodiment, the linked HLA molecules are encoded by HLA class II genes at 3 or more different loci. In another embodiment, the linked HLA molecules are encoded by HLA class II genes at 4 separate loci. In another embodiment, the linked HLA molecules are encoded by HLA class II genes at 4 or more other loci. In another embodiment, the linked HLA molecules are encoded by HLA class II genes at more than 4 different loci. On In other embodiments, the loci are selected from the HLA-DRB loci. In another embodiment, the HLA class II binding peptide is an HLA-DRA binding peptide. In another embodiment, the peptide is an HLA-DQA1 binding peptide. In another embodiment, the peptide is an HLA-DQB 1 binding peptide. In another embodiment, the peptide is an HLA-DPA1 binding peptide. In another embodiment, the peptide is an HLA-DPB 1 binding peptide. In another embodiment, the peptide is an HLA-DMA binding peptide. In another embodiment, the peptide is an HLA-DMB binding peptide. In another embodiment, the peptide is an HLA-DOA binding peptide. In another embodiment, the peptide is an HLA-DOB binding peptide. In another embodiment, the peptide binds to any other HLA class II molecule known in the art.

In another embodiment, a WT1 peptide of the present invention binds 2 different HLA-DRB molecules. In another embodiment, the peptide binds to 3 different HLA-DRB molecules. In another embodiment, the peptide binds to 4 different HLA-DRB molecules. In another embodiment, the peptide binds to 5 different HLA-DRB molecules. In another embodiment, the peptide binds to 6 different HLA-DRB molecules. In another embodiment, the peptide binds to more than 6 different HLA-d Rb molecules.

In another embodiment, a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 different HLA-DRB alleles. In another embodiment, HLA-DRB molecules are encoded by 3 different HLA-DRB alleles. In another embodiment, HLA-DRB molecules are encoded by 4 different h LA-DRB alleles. In another embodiment, HLA-DRB molecules are encoded by 5 different HLA-DRB alleles. In another embodiment, HLA-DRB molecules are encoded by 6 different HLA-DRB alleles. In another embodiment, HLA-DRB molecules are encoded by more than 6 different HLA-DRB alleles.

In another embodiment, a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In Another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 3 different HLA-DRB alleles selected from d Rb 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 4 different HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, DRB 1104, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.

In another embodiment, the present invention provides a composition comprising 2 different WT1 peptides of the present invention. In another embodiment, the 2 distinct WT1 peptides are both unaltered. In another embodiment, 1 of the WT1 peptides is unaltered, while the other is heteroclite. In another embodiment, both WT1 peptides are heteroclites.

In another embodiment, the composition comprises 3 different WT1 peptides of the present invention. In another embodiment, the composition comprises 4 different WT1 peptides of the present invention. In another embodiment, the composition comprises 5 different WT1 peptides of the present invention. In another embodiment, the composition comprises more than 5 different isolated WT1 peptides of the present invention.

In another embodiment, 2 of the WT1 peptides in the composition are unaltered. In another embodiment, 2 of the WT1 peptides in the composition are heteroclites. In another embodiment, 2 of the WT1 peptides in the composition are unaltered and 2 are heteroclites. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered. In another embodiment, more than 2 of the WT1 peptides in the composition are heteroclites. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered and more than 2 are heteroclite.

In another embodiment, 1 of the additional WT1 peptides in a composition of the present invention has a sequence selected from the sequences set forth in SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, 2 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 46, 47, 48, 49, 50 and 55 In another embodiment, 3 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 46, 47, 48, 49, 50 and 55.

In another embodiment, any other immunogenic WT1 peptide known in the art is used as an additional WT1 peptide. In another embodiment, any combination of immunogenic WT1 peptides known in the art is used. Non-limiting sources of other WT1 peptides include WO2005053618, WO2007047764 and WO2007120673.

In another embodiment, a composition of the present invention contains 2 HLA class II heteroclite peptides that are derived from the same isolated WT1 peptide of the present invention. In another embodiment, the 2 HLA class II heteroclite peptides contain mutations at different anchoring residues to HLA class II molecules. In another embodiment, the 2 HLA class II heteroclite peptides contain different mutations in them. anchor residues. In another embodiment, 2 of the HLA class II heteroclite peptides are derived from different isolated WT1 peptides of the present invention.

In another embodiment, 2 WT1 peptides of the present invention, or the WT1 peptides corresponding to two HLA class II heteroclite peptides of the present invention, overlap each other. In another embodiment, the overlap between the peptides is at least 7 amino acids (AA). In another embodiment, the overlap is at least 8 AA. In another embodiment, the overlap is at least 9 AA. In another embodiment, the overlap is 7 AA. In another embodiment, the overlap is 8 AA. In another embodiment, the overlap is 9 AA. In another embodiment, the overlap is 10 AA. In another embodiment, the overlap is 11 AA. In another embodiment, the overlap is 12 AA. In another embodiment, the overlap is 13 AA. In another embodiment, the overlap is 14 AA. In another embodiment, the overlap is 15 AA. In another embodiment, the overlap is 16 AA. In another embodiment, the overlap is more than 16 AA.

In another embodiment, the peptides in a composition of the present invention bind to 2 different HLA class II molecules. In another embodiment, the peptides bind to 3 different HLA class II molecules. In another embodiment, the peptides bind to 4 different HLA class II molecules. In another embodiment, the peptides bind to 5 different HLA class II molecules. In another embodiment, the peptides bind to more than 5 different HLA class II molecules. In another embodiment, the peptides of the composition bind to the same HLA class II molecules.

In another embodiment, each of the WT 1 peptides in a composition of the present invention binds to a pool of HLA class II molecules. In another embodiment, each of the WT1 peptides binds to a different set of HLA class II molecules. In another embodiment, the WT1 peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, 2 of the WT1 peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, 2 or more of the WT1 peptides bind to the same set of HLA class II molecules, while another of the WT1 peptides binds to a different set. In another embodiment, 2 or more of the WT1 peptides bind to an overlapping set of HLA class II molecules, while another of the WT1 peptides binds to a different set.

In another embodiment, 2 or more of the WT1 peptides in a composition of the present invention each bind more than 1 HLA-DRB molecule. In another embodiment, the 4 or more HLA-DRB molecules linked by the peptides in the composition are distinct from each other. In another embodiment, HLA-DRB molecules are encoded by different HLA-DRB alleles.

In another embodiment, 2 or more of the HLA class II molecules linked by WT1 peptides in a composition of the present invention are HLA-DRB molecules. In another embodiment, 3 or more of the HLA class II molecules that bind are HLA-DRB molecules. In other embodiments, the HLA class II molecules that bind can be any of the HLA class II molecules listed in the present disclosure. In another embodiment, the HLA class II molecules that bind are encoded by 2 or more different HLA class II alleles at a given locus. In another embodiment, the binding HLA class II molecules are encoded by HLA class II genes at 2 or more other loci.

In another embodiment, a "set of HLA class II molecules" refers to HLA class II molecules encoded by different alleles at a particular locus. In another embodiment, the term refers to HLA class II molecules with a particular binding specificity. In another embodiment, the term refers to HLA class II molecules with a particular peptide consensus sequence. In another embodiment, the term refers to a superfamily of HLA class II molecules.

In another embodiment, the present invention provides a composition comprising an unaltered HLA class II molecule-binding WT1 peptide of the present invention and a second HLA class I molecule-binding WT1 peptide. In another embodiment, the composition comprises more than 1 HLA class II binding peptide Wt 1 of the present invention, in addition to the HLA class I binding peptide WT1. In another embodiment, the composition comprises more than 1 HLA class I binding peptide WT1 class I, in addition to the HLA class II binding peptide WT1.

In another embodiment, the AA sequence of the other HLA class I binding peptide WT1 comprises a sequence selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, the AA sequence of the other HLA class I molecule binding peptide WT1 is selected from the sequences set forth in SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47 , 48, 49, 50 and 55.

In another embodiment, the HLA class I binding peptide WT1 is a heteroclite HLA class I peptide. In another embodiment, the HLA class I binding peptide WT1 contains a mutation in an anchor residue of the HLA class I molecule thereof, as described later in the present description. As provided herein, WT1 derived peptides were modified at HLA anchoring residues to generate heteroclite peptides with predicted increased binding to HLA-A0201 and HLA-A0301. Peptides with a predicted increase in binding they also showed an improved ability to bind to HLA class I molecules and increased immunogenicity.

In another embodiment, the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I heteroclite peptide. In another embodiment, the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, a substitution at position 1 (eg, to tyrosine) stabilizes the binding of the anchor residue at position 2.

In another embodiment, the mutation is at position 2 of the HLA class I heteroclite peptide. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to methionine. In another embodiment, the residue is changed to any other residue known in the art.

In another embodiment, the mutation is at position 6 of the HLA class I heteroclite peptide. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to glutamine. In another embodiment, the residue is changed to histidine. In another embodiment, the residue is changed to any other residue known in the art.

In another embodiment, the mutation is at position 9 of the HLA class I heteroclite peptide. In another embodiment, the mutation changes the residue at the C-terminal position thereof. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to alanine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to any other residue known in the art.

In another embodiment, the point mutation is in a primary anchor residue. In another embodiment, the primary HLA class I anchor residues are at positions 2 and 9. In another embodiment, the point mutation is at a secondary anchor residue. In another embodiment, the secondary HLA class I anchor residues are at positions 1 and 8. In another embodiment, the secondary HLA class I anchor residues are at positions 1, 3, 6, 7 and 8. In another embodiment, the point mutation is at a position selected from positions 4, 5, and 8.

In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 1, 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is at 1 or more residues in positions selected from positions 1, 3, 6, and 9. In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 1, 2, 6, and 9. In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 1, 6, and 9. In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 1, 2, and 9. In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 1, 3, and 9. In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 2 and 9. In another embodiment, the point mutation is at 1 or more residues at positions selected from positions 6 and 9.

In another embodiment, the HLA class I molecule binding peptide WT is 9 AA in length. In another embodiment, the peptide is 10 AA in length. As provided herein, the native and heteroclite 9-10 AA peptides exhibited substantial binding to HLA class I molecules and the ability to induce cytokine secretion and cytolysis by CTL.

In another embodiment, the HLA class I molecule that is bound by the WT1 peptide that is bound by the HLA class I molecule is an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-A2 molecule. In another embodiment, the HLA class I molecule is an HLA-A3 molecule. In another embodiment, the HLA class I molecule is an HLA-A1 1 molecule. In another embodiment, the HLA class I molecule is an HLA-B 8 molecule. In another embodiment, the HLA class I molecule is an HLA molecule -0201. In another embodiment, the HLA class I molecule binds to any other HLA class I molecule known in the art.

In another embodiment, a WT1 peptide of the present invention is 9-11 amino acids (AA) in length. In another embodiment, the peptide ranges in size from 9-18, or in another embodiment, 9-15, or in another embodiment, 9-11 AA in length. In another embodiment, the peptide is 9 AA or in another embodiment, 10 AA or in another embodiment, 12 AA in length, or in another embodiment, any length in between.

In another embodiment, a peptide of the present invention is a length variant of a peptide listed in the Examples. In another embodiment, the length variant is one amino acid (AA) shorter than the peptide of the Examples. In another embodiment, the length variant is two AA shorter than the peptide of the Examples. In another embodiment, the length variant is more than two AA shorter than the peptide of the Examples. In another embodiment, the shorter peptide is truncated at the N-terminus. In another embodiment, the shorter peptide is truncated at the C-terminus. In another embodiment, the truncated peptide is truncated at both the N-terminus and the C-terminus. Peptides are, in another embodiment, capable of truncation without changing the affinity for HLA molecules, as is well known in the art.

In another embodiment, the length variant is longer than a peptide listed in the Examples of the present invention. In another embodiment, the longest peptide is extended at the N-terminus in accordance with the surrounding WT1 sequence. Peptides are, in another embodiment, susceptible to extension at the N-terminus without changing the affinity for HLA molecules, as is well known in the art. Therefore, said peptides are equivalent to the peptides listed in the examples. In another embodiment, the N-terminal extended peptide is extended by a residue. In another embodiment, the N-terminal extended peptide is spanned by two residues. In another embodiment, the N-terminal extended peptide spans three residues. In another embodiment, the N-terminal extended peptide spans more than three residues.

In another embodiment, the longest peptide is extended at the C-terminus in accordance with the surrounding WT1 sequence. In another embodiment, the peptides are C-terminal extensible without changing the affinity for HLA molecules, as is well known in the art. Therefore, said peptides are equivalents of the peptides listed in the Examples of the present invention. In another embodiment, the C-terminal extended peptide is extended by a residue. In another embodiment, the C-terminal extended peptide is spanned by two residues. In another embodiment, the C-terminal extended peptide spans three residues. In another embodiment, the C-terminal extended peptide spans more than three residues.

In another embodiment, the extended peptide is extended at both the N-terminus and the C-terminus in accordance with the surrounding WT1 sequence.

In another embodiment, a truncated peptide of the present invention retains the HLA anchoring residues (eg, the HLA class I anchoring residues) at the second residue and the C-terminal residue, with fewer intermediate residues. (eg, 5) than a peptide listed in the Examples of the present invention. Peptides are, in another embodiment, susceptible to such a mutation without changing the affinity for HLA molecules. In another embodiment, said truncated peptide is designed by removing one of the intermediate residues from one of the above sequences. In another embodiment, the HLA anchoring residues are retained at the second and eighth residues. In another embodiment, the HLA anchoring residues are retained at the first and eighth residues.

In another embodiment, an extended peptide of the present invention retains the HLA anchoring residues (eg, the HLA class I anchoring residues) at the second residue and the C-terminal residue, with a greater number of intermediate residues. (eg, 7 or 8) than a peptide listed in the Examples of the present invention. In another embodiment, said extended peptide is designed by adding one or more residues between two of the intermediate residues of one of the above sequences. It is well known in the art that residues can be removed or added between the intermediate HLA-binding peptide sequences without changing the affinity for HLA. Therefore, said peptides are equivalents of the peptides listed in the Examples of the present invention. In another embodiment, the HLA anchoring residues are retained at the second and ninth residues. In another embodiment, the HLA anchoring residues are retained at the first and eighth residues. In another embodiment, the HLA anchoring residues are retained at the two residues separated by six intermediate residues.

"Fragment", in another embodiment, refers to a peptide 11 or more AA in length. In another embodiment, a peptide of the present invention is 16 Aa in length. In another embodiment, the peptide is 12 AA in length. In another embodiment, the peptide is 13 AA. In another embodiment, the peptide is 14 AA. In another embodiment, the peptide is 15 or more AA. In another embodiment, the peptide is 17 AA.

In another embodiment, the present invention provides a composition comprising a peptide of this invention. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the composition further comprises an adjuvant. In another embodiment, the composition comprises 2 or more peptides of the present invention. In another embodiment, the composition further comprises any of the additives, compounds, or excipients set forth below. In another embodiment, the adjuvant is KLH, QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, or alum. In other embodiments, the carrier is any carrier listed in the present description. In other embodiments, the adjuvant is any adjuvant listed in the present disclosure.

In another embodiment, this invention provides a vaccine comprising a peptide of this invention. In another embodiment, this invention provides a vaccine comprising an antigen presenting cell (APC) and a peptide of this invention. In another embodiment, the vaccine further comprises a carrier. In another embodiment, the vaccine further comprises an adjuvant. In another embodiment, the vaccine further comprises an APC. In another embodiment, the vaccine further comprises a combination of more than 1 of an antigen, carrier, and / or APC. In another embodiment, the vaccine is a cell-based composition.

In another embodiment, the term "vaccine" refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response to a particular disease, condition, or symptom thereof. In another embodiment, this invention encompasses peptide-based vaccines, wherein the peptide comprises any embodiment listed in the present disclosure, including immunomodulatory compounds such as cytokines, adjuvants, etc.

In another embodiment, a vaccine of the present invention further comprises an adjuvant. In another embodiment, the adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. In another embodiment, the adjuvant is GM-CSF. Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APCs, and dendritic cells and T cells.

In another embodiment, the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum.

In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the WT1 vaccine comprises two of the foregoing adjuvants. In another embodiment, the WT1 vaccine comprises more than two of the foregoing adjuvants.

In other embodiments, a vaccine or composition of the present invention may comprise any of the WT1 peptide embodiments of the present invention and combinations thereof.

It should be understood that any of the embodiments described in the present disclosure, with respect to peptides, vaccines, and compositions of this invention, may be employed in any of the medical uses of this invention.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in treating a subject with a WT1-expressing cancer.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in treating a subject with an MDS.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in suppressing or arresting the progression of a WT1 expressing cancer.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in reducing the incidence of a WT1-expressing cancer in a subject.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in reducing the incidence of AML.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in reducing the incidence of relapse of a WT1-expressing cancer in a subject.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in reducing the incidence of relapse of AML in a subject.

In another embodiment, the present invention provides a WT1 vaccine of the present invention for use in disrupting T cell tolerance to a WT1 expressing cancer.

In another embodiment, the present application describes a method of treating a subject having a WT1-expressing cancer, comprising (a) inducing in a donor the formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method described in the present description; and (b) infusing the human CTL into the subject, and thereby treating a subject having cancer.

In another embodiment, the present application describes a method of treating a subject having a WT 1-expressing cancer, comprising (a) inducing the ex vivo formation and proliferation of human CTLs that recognize a malignant cancer cell by a method described. in the present description, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, and thereby treating a subject having cancer.

Methods for ex vivo immunotherapy are well known in the art and are described, for example, in US Patent Application Nos. Serial 2006/0057130, 2005/0221481, 2005/0214268, 2003/0175272, 2002/0127718 and US Patent No. 5,229,115. Additional methods are well known in the art and are described, for example, in Davis ID et al., (Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8 + T cells efficiently in cancer patients. J Immunother. 2006 Sep-Oct; 29 (5): 499-511) and Mitchell MS et al., (The cytotoxic T cell response to peptide analogs of the HLA-A * 0201 - restricted MUC1 signal sequence epitope, M1.2. Cancer Immunol Immunother.

July 28, 2006).

In another embodiment, the present application describes a method for inducing the formation and proliferation of CTL specific for WT1-expressing cancer cells, the method comprises contacting a population of lymphocytes with a vaccine of the present invention. In another embodiment, the vaccine is an APC associated with a peptide of the present invention. In another embodiment, the vaccine is an APC associated with a mixture of peptides of the present invention.

Therefore, in another embodiment, the present invention provides a peptide of the invention or a nucleic acid molecule encoding the peptide or a vector comprising the nucleic acid molecule to induce in vitro the formation and proliferation of cell-specific CTLs. of a cancer that expresses WT1.

In another embodiment, this invention provides a vaccine of the present invention for use in generating a heteroclite immune response directed against a WT1-expressing cancer in a subject.

In another embodiment, the present invention provides an immunogenic composition comprising a peptide of the invention for use in inducing an anti-mesothelioma immune response in a subject. In another embodiment, mesothelioma is a malignant mesothelioma.

In another embodiment, the present invention provides an immunogenic composition comprising a nucleotide molecule encoding a peptide of the invention for use in inducing an anti-mesothelioma immune response in a subject. In another embodiment, mesothelioma is a malignant mesothelioma.

In another embodiment, the present invention provides an immunogenic composition comprising a peptide of the invention for use in treating a subject with a mesothelioma. In another embodiment, mesothelioma is a malignant mesothelioma.

In another embodiment, the present invention provides an immunogenic composition comprising a nucleotide molecule encoding a peptide of the invention for use in treating a subject with mesothelioma. In another embodiment, mesothelioma is a malignant mesothelioma.

In another embodiment, the present invention provides an immunogenic composition comprising a peptide of the invention for use in reducing the incidence of mesothelioma, or its relapse, in a subject. In another embodiment, mesothelioma is a malignant mesothelioma.

In another embodiment, the present invention provides an immunogenic composition comprising a nucleotide molecule encoding a peptide of the invention for use in reducing the incidence of mesothelioma, or its relapse, in a subject. In another embodiment, mesothelioma is a malignant mesothelioma.

In another embodiment, a cell targeted for an immune response elicited by a peptide of the present invention displays the WT1 peptide of the present invention, or a corresponding WT1 fragment, in an HLA molecule. In another embodiment, the HLA molecule is an HLA class I molecule. In other embodiments, the HLA molecule is any HLA class I subtype or HLA class I molecule known in the art. In another embodiment, the immune response against the WT1 peptide or fragment is a heteroclite immune response.

In another embodiment, the WT1-expressing cancer is acute myelogenous leukemia (AML). In another embodiment, WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematologic cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is gastric cancer. In another embodiment, the WT1-expressing cancer is colon cancer. In another embodiment, the WT1-expressing cancer is lung cancer. In another embodiment, the WT1-expressing cancer is breast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is ovarian cancer. In another embodiment, the WT1-expressing cancer is uterine cancer. In another embodiment, the cancer expressing WT 1 is thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is thyroid cancer. In another embodiment, the WT1-expressing cancer is liver cancer. In another embodiment, the WT1-expressing cancer is kidney cancer. In another embodiment, the WT1-expressing cancer is Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.

In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with lung cancer. In another embodiment, the solid tumor is associated with breast cancer. In another embodiment, the solid tumor is associated with colorectal cancer. In another embodiment, the solid tumor is associated with prostate cancer. In another embodiment, the solid tumor is associated with ovarian cancer. On Another modality, the solid tumor is associated with kidney cancer. In another embodiment, the solid tumor is associated with pancreatic cancer. In another embodiment, the solid tumor is associated with brain cancer. In another embodiment, the solid tumor is associated with gastrointestinal cancer. In another embodiment, the solid tumor is associated with skin cancer. In another embodiment, the solid tumor is associated with a melanoma.

In another embodiment, a cancer or tumor to be treated by the present invention is suspected of expressing WT1. In another embodiment, WT1 expression has not been verified by testing the actual tumor sample. In another embodiment, the cancer or tumor is of a type known to express WT1 in many cases. In another embodiment, the type expresses WT1 in most cases.

Any of the modalities listed in the present disclosure, with respect to peptides, vaccines, and compositions of this invention, can be employed in any of the medical uses of this invention.

In another embodiment, multiple peptides of this invention are used to stimulate an immune response in the methods of the present invention.

Those skilled in the art will understand that the methods described in the present disclosure allow the design of other peptides derived from WT1. The methods further allow the design of peptides that bind to other HLA molecules. The methods further allow the design of vaccines combining WT1-derived peptides of the present invention.

In another embodiment, the vaccines of the present invention have the advantage of activating or eliciting WT1-specific CD4 <+> T cells that contain a variety of different HLA class II alleles. In another embodiment, vaccines have the advantage of activating or eliciting WT1-specific CD4 <+> T cells in a substantial proportion of the population (e.g., in different modalities, 50%, 55%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95% or greater than 95%). In another embodiment, the vaccines activate or elicit WT1-specific CD4 <+> T cells in a substantial proportion of a particular population (eg, Caucasian Americans).

In another embodiment, the treatments of the present invention provide an enhancement in an immune response that has already been mounted by a subject. In another embodiment, the treatments of the present invention comprise administering the peptide, composition, or vaccine 2 or more times. In another embodiment, the peptides vary in their composition, concentration, or a combination thereof. In another embodiment, the peptides provide the initiation of an immune response against an antigen of interest in a subject who has not yet initiated an immune response against the antigen. In another embodiment, the CTLs that are induced proliferate in response to presentation of the peptide on the APC or cancer cell. In other embodiments, reference to modulation of the immune response implies, one or both of the humoral and cellular arms of the immune system, which is accompanied by the presence of Th2 and Th1 helper T cells, respectively, or in another embodiment, each arm individually.

In other modalities, treatments that affect tumor growth result in either (1) direct inhibition of tumor cell division, or (2) immune cell-mediated lysis of tumor cells, or both, which leads to a suppression of the net expansion of tumor cells.

Inhibition of tumor growth by either of these two mechanisms can be readily determined by one of skill in the art based on several well-known methods. In another embodiment, tumor inhibition is determined by measuring the actual size of the tumor over a period of time. In another embodiment, tumor inhibition can be determined by estimating the size of a tumor (over a period of time) using methods well known to those of skill in the art. More specifically, a variety of radiological imaging methods can be used (eg, single photon positron emission computed tomography; see generally, "Nuclear Medicine in Clinical Oncology", Winkler, C. (ed.) Springer- Verlag, New York, 1986), to estimate tumor size. Such methods can also use a variety of imaging agents, including, for example, conventional imaging agents (eg, gallium-67 citrate), as well as specialized reagents for imaging metabolites. , receptor imaging or immunological imaging (eg, radiolabeled monoclonal antibody specific tumor markers). In addition, non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984) can also be used to estimate tumor size.

In addition to the in vivo methods for determining tumor inhibition discussed above, a variety of in vitro methods can be used to predict tumor inhibition in vivo. Representative examples include lymphocyte-mediated antitumor cytolytic activity determined, eg, by <51> Cr release assay (Examples), tumor-dependent proliferation of lymphocytes (Ioannides, et al., J. Immunol. 146 (5) : 1700-1707, 1991), in vitro generation of tumor-specific antibodies (Herlyn, et al., J. Immunol. Meth. 73: 157-167, 1984), inhibition of in vitro cell-mediated cell growth (eg, CTL, T helper cells) or humoral (e.g., antibodies) (Gazit, et al., Cancer Immunol Immunother 35: 135-144, 1992) and, for either of these assays, determination of the cell precursor frequency (Vose, Int. J. Cancer 30: 135-142 (1982), and others.

In another embodiment, methods for suppressing tumor growth indicate a growth state that is reduced compared to growth without contact with, or exposure to, a peptide of this invention. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells proliferate using a <3> incorporation assay. H-thymidine or by counting tumor cells. "Suppressing" tumor cell growth refers, in other embodiments, to slowing, delaying or stopping tumor growth or tumor shrinkage.

In another embodiment, WT1 expression is measured. In another embodiment, the expression of the WT1 transcript is measured. In another embodiment, the levels of WT1 protein in the tumor are measured.

Methods for determining the presence and magnitude of an immune response are well known in the art. In another embodiment, lymphocyte proliferation assays, wherein T cell uptake of a radioactive substance, eg, <3> H-thymidine, is measured as a function of cell proliferation. In other modalities, detection of T cell proliferation is achieved by measuring increases in interleukin-2 (IL-2) production, Ca <2+> flux, or dye uptake, such as 3 - (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium.

In another embodiment, CTL stimulation is determined by means known to those of skill in the art, including, detection of cell proliferation, cytokine production, and others. Analysis of the types and amounts of cytokines secreted by T cells upon contact with ligand-pulsed targets can be a measure of functional activity. Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. (Fujihashi K. et al., (1993) J. Immunol. Meth. 160: 181; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13: 259).

In another embodiment, CTL activity is determined by a <51> Cr release lysis assay. Lysis of peptide-pulsed <51> Cr-labeled targets by antigen-specific T cells can be compared to control peptide-pulsed target cells. In another embodiment, T cells are stimulated with a peptide of this invention and lysis of target cells expressing the native peptide can be determined in the context of the MHC. The kinetics of lysis, as well as the overall lysis of the target at a set time point (eg, 4 hours) are used, in another embodiment, to evaluate the performance of the ligand. (Ware C.F. et al., (1983) J Immunol 131: 1312).

Methods for determining the affinity of a peptide for an HLA molecule are well known in the art. In another embodiment, the affinity is determined by TAP stabilization assays.

In another embodiment, the affinity is determined by a competitive radioimmunoassay. In another embodiment, the following protocol is used: Target cells are washed twice in PBS with 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, NJ). The cells are resuspended at 10 <7> / ml on ice and the native peptides bound to the cell surface are separated for 2 minutes at 0 [deg.] C using citrate-phosphate buffer in the presence of 3 mg / ml of beta2 microglobulin. The pellet is resuspended at 5 x 10 <6> cells / ml in PBS / 1% BSA in the presence of 3 mg / ml beta2-microglobulin and 30 mg / ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence. of HLA-specific peptides for 10 minutes at 20 <0> C, then with <125> I-labeled peptide for 30 minutes at 20 <0> C. Total bound <125> I is determined after two washes with PBS / 2% BSA and one wash with PBS. Relative affinities are determined by comparing increasing concentrations of the test peptide against a known binding peptide.

In another embodiment, a specificity analysis of the peptide binding to HLA is performed on the surface of living cells (eg, SKLY-16 cells) to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction. This includes, in another embodiment, competition with excess unlabeled peptides that are known to bind to the same or different HLA molecules and the use of target cells expressing the same or different types of HLA. This assay is performed, in another embodiment, on fresh live human PBMC or 0.25% paraformaldehyde fixed, leukemia cell lines and EBV transformed T cell lines of specific h La types. The relative avidity of peptides found to bind to MHC molecules in specific cells is analyzed by competition assays as described above against known high affinity <125> I-labeled peptides for the relevant HLA molecule, for example , tyrosinase or peptide sequence of HBV. In another embodiment, an HLA class II binding peptide of the methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA. In another embodiment, increasing the length of the HLA class II binding peptide allows binding to more than one HLA class II molecule. In another embodiment, the increase in length allows binding to an HLA class II molecule whose binding motif is not known. In another embodiment, increasing the length allows binding to an HLA class I molecule. In another embodiment, the binding motif of the HLA class I molecule is known. In another embodiment, the binding motif of the HLA class I molecule is not known. the HLA class I molecule.

In another embodiment, the peptides of the present invention comprise a non-classical amino acid such as: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., (1991) J. Am Chem. Soc. 113: 2275- 2283); (2S, 3S) -methylphenylalanine, (2S, 3R) -methylphenylalanine, (2R, 3S) -methyl-phenylalanine and (2R, 3R) -methyl-phenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett. 32 (41): 5769-5772); 2-Aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D. Thesis, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., (1984) J. Takeda Res. Labs. 43: 53-76); Histidine-isoquinoline-carboxylic acid (Zechel et al., (1991) Int. J. Pep. Protein Res.

38 (2): 131-138); and HIC (histidine cycle urea), (Dharanipragada et al., (1993) Int. J. Pep. Protein Res. 42 (1): 68-77) and ((1992) Acta. Crst., Crystal Struc. Comm. 48 (IV): 1239-124).

In another embodiment, a peptide of this invention comprises an AA or peptidomimetic analog that, in other embodiments, induces or favors specific secondary structures. Such peptides comprise, in other embodiments, the following: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a [beta] -turn inducing dipeptide analog (Kemp et al., (1985) J Org. Chem. 50: 5834-5838); [beta] -sheet inducing analogs (Kemp et al., (1988) Tetrahedron Lett. 29: 5081-5082); [beta] -turn inducing analogs (Kemp et al., (1988) Tetrahedron Lett.

29: 5057-5060); alpha helix inducing analogs (Kemp et al., (1988) Tetrahedron Lett. 29: 4935-4938); gamma spin inducing analogs (Kemp et al., (1989) J. Org. Chem. 54: 109: 115); analogs provided by the following references: Nagai and Sato (1985) Tetrahedron Lett. 26: 647-650; and DiMaio et al., (1989) J. Chem. Soc. Perkin Trans, p. 1687; a spin analog of Gly-Ala (Kahn et al., (1989) Tetrahedron Lett. 30: 2317); isosterone amide bond (Jones et al., (1988) Tetrahedron Lett. 29 (31): 3853-3856); tretrazole (Zabrocki et al., (1988) J. Am. Chem. Soc. 110: 5875-5880); DTC (Samanen et al., (1990) Int. J. Protein Pep. Res. 35: 501: 509); and analogs taught in Olson et al., (1990) J. Am. Chem. Sci. 112: 323-333 and Garvey et al., (1990) J. Org. Chem. 55 (3): 936-940. Conformationally constrained mimetics of beta turns and beta bulges, and peptides containing them, are described in US Pat. 5,440,013, issued August 8, 1995 to Kahn.

In other embodiments, a peptide of this invention is conjugated to one of several other molecules, as described below, which may be through covalent or non-covalent bonding (complex formation), the nature of which varies, in another embodiment, depending on the particular purpose. In another embodiment, the peptide forms a complex covalently or non-covalently with a macromolecular carrier (eg, an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinylpyrrolidone and lipids. In another embodiment, a peptide of this invention is bound to a substrate. In another embodiment, the peptide is conjugated to a fatty acid, for introduction into a liposome (US Patent No. 5,837,249). In another embodiment, a peptide of the invention forms a complex covalently or non-covalently with a solid support, a variety of which are known in the art. In another embodiment, binding of the peptide to the carrier, substrate, fatty acid, or solid support serves to enhance an elicited immune response.

In other embodiments, the carrier is thyroglobulin, an albumin (eg, human serum albumin), tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein , keyhole limpet hemocyanin, an albumin or other carrier protein or carrier peptide; recombinant hepatitis B virus vaccine, or an APC.

In another embodiment, the term "amino acid" (AA) refers to a natural AA or, in another embodiment, a non-natural or synthetic AA, and may include, in other embodiments, glycine, D or L optical isomers, AA analogs , peptidomimetics, or combinations thereof.

In another embodiment, the terms "cancer", "neoplasia", "neoplastic" or "tumor" are used interchangeably and refer to cells that have undergone a malignant transformation that renders them pathological to the host organism. Primary cancer cells (ie, cells obtained near the site of malignant transformation) can be easily distinguished from non-cancer cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from an ancestor of the cancer cell. This includes metastasized cancer cells and in vitro cultures and cell lines derived from cancer cells. In another embodiment, a tumor is detectable on the basis of tumor mass; for example, by procedures such as computerized axial tomography, magnetic resonance imaging (MRI), x-ray, ultrasound or palpation, and in another modality, it is identified by biochemical or immunological findings, the latter being used to identify cancer cells , also, in other modalities.

Methods for synthesizing peptides are well known in the art. In another embodiment, the peptides of this invention are synthesized using an appropriate solid state synthesis procedure (see, for example, Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, California (1968); Merrifield ( 1967) Recent Progress in Hormone Res 23: 451). The activity of these peptides is tested, in other embodiments, using assays as described herein.

In another embodiment, the peptides of this invention are purified by standard methods, including chromatography (eg, ion exchange, affinity, and size exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for purification of proteins. In another embodiment, immunoaffinity chromatography is used, in which an epitope is isolated by binding to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and attached to a stationary support. .

In another embodiment, affinity tags such as hexa-His (Invitrogen), maltose-binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al., (1991) Meth. Enzymol. 194: 508-509 ), glutathione-S-transferase, or others, bind to the peptides of this invention to allow easy purification by passage over an appropriate affinity column. Isolated peptides can also be physically characterized, in other embodiments, using techniques such as proteolysis, nuclear magnetic resonance, and x-ray crystallography.

In another embodiment, the peptides of this invention are produced by in vitro translation, by known techniques, as will be apparent to one of ordinary skill in the art. In another embodiment, peptides are differentially modified during or after translation, for example, by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, binding to an antibody molecule, membrane molecule, or other ligand, (Ferguson et al., (1988) Ann. Rev. Biochem. 57: 285-320).

In another embodiment, the peptides of this invention further comprise a detectable label, which in another embodiment is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense. In other embodiments, the detectable marker comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase, <32> P, <125> I , <3> H and <14> C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other markers known to one skilled in the art. The particular marker used will depend on the type of immunoassay used.

In another embodiment, a peptide of this invention binds to a substrate that, in another embodiment, serves as a carrier. In another embodiment, binding of the peptide to a substrate serves to enhance an elicited immune response.

In another embodiment, the peptides of this invention are attached to other molecules, as described herein, through the use of conventional cross-linking agents such as carbodiimides. Examples of carbodiimides are 1-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide (CMC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 1-ethyl-3- (4- azonia-44-dimethylpentyl) carbodiimide.

In other embodiments, the crosslinking agents include cyanogen bromide, glutaraldehyde, and succinic anhydride. In general, any of a number of homo-bifunctional agents can be used, including a homobifunctional aldehyde, a homobifunctional epoxide, a homobifunctional imido-ester, a homobifunctional N-hydroxysuccinimide ester, a homobifunctional maleimide, a homobifunctional alkyl halide, a homobifunctional pyridyl disulfide, a homobifunctional aryl halide, a homobifunctional hydrazide, a homobifunctional diazonium derivative, and a homobifunctional photoreactive compound. Also envisioned, in other embodiments, are heterobifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group, and compounds with a carbonyl-reactive and a reactive group. sulfhydryl.

In other embodiments, homo-bifunctional crosslinking agents include the bifunctional N-hydroxysuccinimide esters dithiobis (succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartrate; the bifunctional imidoesters dimethyl adipimidate, dimethyl pimellimidate and dimethyl suberimidate; the sulfhydryl reactive bifunctional crosslinkers 1,4-di- [3 '- (2'-pyridyldithio) propionamido] butane, bismaleimidohexane and bis-N-maleimido-1,8-octane; the bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis- [b- (4-azidosalicylamido) ethyl] disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde and adipaldehyde; a bifunctional epoxide such as 1,4-butanediol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bisdiazotized benzidine; the bifunctional alkyl halides N1N'-ethylene-bis (iodoacetamide), N1N'-hexamethylene-bis (iodoacetamide), N1N'-undecamethylenebis (iodoacetamide), as well as benzyl halides and halomostases, such as ala'-diiodo acid- p-xylene sulfonic and tri (2-chloroethyl) amine, respectively,

In other embodiments, heterobifunctional crosslinking agents used to attach peptides to other molecules, as described herein, include, but are not limited to, SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate ), MB S (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl (4-iodoacteyl) aminobenzoate), SMPB (succinimidyl-4- (p-maleimidophenyl) butyrate), GMBS (N- (ester gamma-maleimidobutyryloxy) succinimide), MPBH (4- (4-N-maleimidophenyl) butyric acid hydrazide), M2C2H (4- (N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl- a- (2-pyridyldithio) toluene) and SPDP (N-succinimidyl 3- (2-pyridyldithio) propionate).

In another embodiment, the peptides of the invention are formulated as non-covalent bonding of monomers through ionic, adsorptive, or biospecific interactions. Peptide complexes with highly positively or negatively charged molecules can be achieved, in another embodiment, by the formation of salt bridges in low ionic strength environments, such as in deionized water. Large complexes can be created, in another embodiment, by using charged polymers such as poly (L-glutamic acid) or poly (L-lysine), which contain numerous negative and positive charges, respectively. In another embodiment, the peptides are adsorbed to surfaces such as microparticle beads of latex or other hydrophobic polymers, by forming non-covalently associated superantigen peptide complexes that effectively mimic chemically polymerized or cross-linked proteins, in other embodiments. In another embodiment, peptides are non-covalently bound to through the use of biospecific interactions between other molecules. For example, utilization of biotin's strong affinity for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes. Peptides, in accordance with this aspect, and in another embodiment, can be modified to possess biotin groups through the use of common biotinylation reagents such as D-biotin N-hydroxysuccinimidyl ester (NHS-biotin), which reacts with amine groups. available.

In another embodiment, a peptide of the present invention is bound to a carrier. In another embodiment, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), influenza, protein Hepatitis B virus core, Hepatitis B virus recombinant vaccine, and the like.

In another embodiment, the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated on a bead.

In another embodiment, the compositions of this invention further comprise immunomodulatory compounds. In other embodiments, the immunomodulatory compound is a cytokine, chemokine, or complement component that enhances the expression of accessory or adhesion molecules of the immune system, their receptors, or combinations thereof. In some embodiments, the immunomodulatory compound includes interleukins, eg, interleukins 1 to 15, interferons alpha, beta, or gamma, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage activating and chemotactic factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia and MIP -Ib, complement components or combinations thereof. In other embodiments, the immunomodulatory compound stimulates the expression or enhances the expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or a combination of them.

In another embodiment, the immunomodulatory compound induces or enhances the expression of costimulatory molecules that participate in the immune response, including, in some embodiments, CD40 or its ligand, CD28, CTLA-4, or a B7 molecule. In another embodiment, the immunomodulatory compound induces or enhances the expression of a thermostable antigen (HSA) (Liu Y. et al., (1992) J. Exp. Med. 175: 437-445), sulfate-modified non-variable chain of MHC of chondroitin (Ii-CS) (Naujokas MF et al., (1993) Cell 74: 257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van RH (1992) Cell 71: 1065-1068), which, in another embodiment, helps joint stimulation by interacting with its cognate ligands on T cells.

In another embodiment, the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents, and the like. In another embodiment, the solvent is an aqueous isotonic buffered solution with a pH of about 7.0. In another embodiment, the composition comprises a diluent such as water, phosphate buffered saline, or saline. In another embodiment, the composition comprises a solvent, which is not aqueous, such as propylethylene glycol, polyethylene glycol, and vegetable oils.

In another embodiment, the composition is formulated for administration by any of the many techniques known to those of skill in the art. For example, this invention provides for parenteral, intravenous, subcutaneous, intradermal, intramucosal, topical, oral, or inhalation administration of the pharmaceutical composition.

In another embodiment, the vaccine comprising a peptide of this invention further comprises a cell population that, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells, or combinations thereof, which, in another embodiment , are autologous, syngeneic or allogeneic, to each other. In another embodiment, the cell population comprises a peptide of the present invention. In another embodiment, the cell population takes up the peptide.

In another embodiment, the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukapheresis blood products, apheresis blood products, peripheral lymph nodes, lymphoid tissue associated with intestine, spleen, thymus, umbilical cord blood, mesenteric lymph nodes, liver, sites of immune injury, eg, synovial fluid, pancreas, cerebrospinal fluid, tumor specimens, granulomatous tissue, or any other source from which such cells can be obtained. In another embodiment, cell populations are derived from human sources, which are, in other embodiments, from neonatal, infant, or adult human sources. In another embodiment, the cell populations of this invention are obtained from animal sources, such as, for example, swine or apes, or any other animal of interest. In another embodiment, the cell populations of this invention are obtained from subjects who are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest.

In another embodiment, cell populations are separated by affinity-based separation methods. Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents attached to a monoclonal antibody or used in conjunction with a monoclonal antibody, eg, complement, and cytotoxins and "immunoadsorption" with an antibody bound to a solid matrix, such as a plate, or any other convenient technique. In another embodiment, the separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detector channels, impedance, etc. In other embodiments, any technique that allows separation of the cell populations of this invention may be employed and should be considered as part of this invention.

In another embodiment, dendritic cells are from the various populations of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, labeled as such (Steinman (1991) Ann. Rev. Immunol. 9: 271-296 ). In another embodiment, the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, isolated from / derived from peripheral blood, or in another embodiment, they are derived from, or are a cell line.

In another embodiment, the cell populations described in the present disclosure are isolated from the white blood cell fraction of a mammal, such as a murine, an ape, or a human (see, for example, WO 96/23060). . The white blood cell fraction can, in another embodiment, be isolated from the peripheral blood of the mammal.

Methods for isolating dendritic cells are well known in the art. In another embodiment, DCs are isolated by a method that includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukaphoresis; (b) separating the white blood cell fraction from step (a) into four or more sub-fractions by countercurrent centrifugal elutriation; (c) stimulate the conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, ( d) identifying the dendritic cell enriched fraction from step (c); and (e) collecting the enriched fraction from step (d), preferably at about 4 [deg.] C.

In another embodiment, the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2 and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57 and CD 19, 20.

In another embodiment, the cell population comprises lymphocytes, which are, in another embodiment, T cells, or in another embodiment, B cells. T cells are characterized, in other embodiments, as NK cells, helper T cells, cytotoxic T lymphocytes ( CTL), TBL, naïve T cells, or combinations thereof. It should be understood that T cells that are primary, or cell lines, clones, etc. should be considered as part of this invention. In another embodiment, the T cells are CTL, or CTL lines, CTL clones, or CTL isolated from tumors, inflammatory, or other infiltrates.

In another embodiment, hematopoietic stem cells or early progenitor cells comprise the cell populations used in this invention. In another embodiment, these populations are isolated or derived by leukapheresis. In another embodiment, leukapheresis follows the administration of cytokines, bone marrow, peripheral blood (PB), or neonatal umbilical cord blood. In another embodiment, stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34 <+>, and the exclusion of the expression of the surface lineage antigen markers, Lin-.

In another embodiment, a peptide, composition, or vaccine of this invention will be administered to a subject in conjunction with bone marrow cells. In another embodiment, the mode of administration in conjunction with bone marrow cells follows prior irradiation of the subject, as part of the course of therapy, to suppress, inhibit, or treat cancer in the subject.

In another embodiment, the phrase "contacting a cell" or "contacting a population" refers to a method of exposure, which may be, in other embodiments, direct or indirect. In another embodiment, said contacting comprises direct injection of the cell through any means well known in the art, such as microinjection. It is also envisioned, in another embodiment, that delivery to the cell is indirect, such as through delivery in a culture medium surrounding the cell, or administration to a subject, via any route well known in the art, and as described in the present description.

In another embodiment, the generation of CTL described in the present disclosure is achieved in vivo and effected by introducing into a subject an antigen-presenting cell that is contacted in vitro with a peptide of this invention (see, for example , Paglia et al., (1996) J. Exp. Med. 183: 317-322).

In another embodiment, the peptides and compositions of the present invention are delivered to the APC. In another embodiment, the peptide-pulsed APCs will be administered to a subject to elicit an immune response or to treat or inhibit the growth or recurrence of a tumor.

In another embodiment, the peptides are delivered to the APC in the form of cDNA encoding the peptides. In another embodiment, the term "antigen presenting cells" (APC) refers to dendritic cells (DC), monocytes / macrophages, B lymphocytes, or other cell type (s) that express the necessary costimulatory / MHC molecules, that efficiently allow T-cell recognition of the displayed peptide. In another embodiment, the APC is a cancer cell.

In another embodiment, the CTLs are contacted with 2 or more APC populations. In another embodiment, the 2 or more APC populations present different peptides.

In another embodiment, techniques that lead to antigen expression in the APC cytosol (eg, DC) are used to deliver the peptides to the APC. Methods for expressing antigens on APC are well known in the art. In another embodiment, techniques include (1) introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of the APC with recombinant vectors expressing a peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC through the use of liposomes. (See Boczkowski D. et al., (1996) J. Exp. Med. 184: 465-472; Rouse et al. (1994) J. Virol. 68: 5685-5689; and Nair et al. (1992) J. Exp. Med. 175: 609-612).

In another embodiment, promoter APCs are used such as those derived from the human cell line 174xCEM.T2, designated T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with MHC class molecules. I from the cell surface (Zweerink et al., (1993) J. Immunol.

150: 1763-1771), as exemplified in the present description.

In another embodiment, as described herein, the subject is exposed to a peptide, or a composition / cell population comprising a peptide of this invention, that differs from the native expressed protein, wherein an immune response is subsequently developed. of the host that cross-reacts with the native protein / antigen.

In another embodiment, the subject, as mentioned in any of the methods or embodiments of this invention, is a human. In other embodiments, the subject is a mammal, which can be a mouse, rat, rabbit, hamster, guinea pig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape.

In another embodiment, the peptides, vaccines, and compositions of this invention stimulate an immune response that results in the lysis of tumor cells.

In another embodiment, any of the methods described in the present description are used to elicit CTL, which are elicited in vitro. In another embodiment, CTLs are elicited ex vivo. In another embodiment, CTLs are elicited in vitro. The resulting CTLs, in another embodiment, will be administered to the subject, thereby treating the condition associated with the peptide, an expression product comprising the peptide, or a homologue thereof.

In another embodiment, the described method involves the introduction of the genetic sequence encoding the peptides of this invention by using, for example, one or more nucleic acid delivery techniques. The nucleic acids of the invention include, in another embodiment, DNA, RNA, and mixtures of DNA and RNA, alone or together with components that are not nucleic acids. In another embodiment, the described method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R. W. et al., Virology (1994) 200: 54). In another embodiment, the disclosed method comprises administering to the subject naked DNA encoding a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al., PNAS-USA (1990) 90: 11307). In another embodiment, analogue- and multi-epitope-based cancer vaccines are used (Fikes et al., Design of multi-epitope, analogue-based cancer vaccines. Expert Opin Biol Ther., 2003 Sep; 3 (6): 985-93 ).

Nucleic acids can be administered to a subject by any means known in the art, including parenteral or intravenous administration, or in other modality, by means of a gene gun. In another embodiment, the nucleic acids will be administered in a composition, corresponding, in other embodiments, to any embodiment listed in the present disclosure.

Vectors for use in accordance with this invention may comprise any vector that facilitates or enables expression of a peptide of this invention. Vectors comprise, in some embodiments, attenuated viruses, such as vaccinia or fowlpox, as described in, for example, US Pat. 4,722,848. In another embodiment, the vector is BCG (Bacillus Calmette Guerin), as described in Stover et al., (Nature 351: 456-460 (1991)). A wide variety of other vectors useful for the therapeutic administration or immunization of the peptides of the invention, for example, Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

In another embodiment, the vector further encodes an immunomodulatory compound, as described herein. In another embodiment, an additional vector encoding the same will be administered to the subject, simultaneously, before or after administration of the vector encoding a peptide of this invention to the subject.

In another embodiment, the peptides, compositions, and vaccines of this invention will be administered to a subject, or used in the treatments of this invention, in combination with other anticancer and chemotherapeutic compounds, including monoclonal antibodies directed against alternative cancer antigens. , or in another embodiment, epitopes consisting of an AA sequence corresponding to, or in part to, that from which the peptides of this invention are derived.

This invention contemplates various modalities of dose ranges. [mu] refers to micro; [mu] g refers to microgram or micrograms. In another embodiment, the dose is 20 µg per peptide per day. In another embodiment, the dose is

dosis es 1500 [mu]g/péptido/día. En otra modalidad, la dosis es 2000 [mu]g/péptido/día.10 µg / peptide / day. In another embodiment, the dose is 30 µg / peptide / day. In another embodiment, the dose is 40 µg / peptide / day. In another embodiment, the dose is 60 µg / peptide / day. In another embodiment, the dose is 80 µg / peptide / day. In another embodiment, dose is 100 µg / peptide / day. In another embodiment, the dose is 150 µg / peptide / day. In another embodiment, dose is 200 µg / peptide / day. In another embodiment, the dose is 300 µg / peptide / day. In another embodiment, dose is 400 µg / peptide / day. In another embodiment, the dose is 600 µg / peptide / day. In another embodiment, dose is 800 µg / peptide / day. In another embodiment, the dose is 1000 µg / peptide / day. In another mode,

dose is 1500 µg / peptide / day. In another embodiment, the dose is 2000 µg / peptide / day.

In another embodiment, the dose is 10 µg / peptide / dose. In another embodiment, the dose is 30 µg / peptide / dose. In another embodiment, the dose is 40 µg / peptide / dose. In another embodiment, the dose is 60 µg / peptide / dose. In another embodiment, the dose is 80 µg / peptide / dose. In another embodiment, the dose is 100 µg / peptide / dose. In another embodiment, the dose is 150 µg / peptide / dose. In another embodiment, the dose is 200 µg / peptide / dose. In another embodiment, the dose is 300 µg / peptide / dose. In another embodiment, the dose is 400 µg / peptide / dose. In another embodiment, the dose is 600 µg / peptide / dose. In another embodiment, the dose is 800 µg / peptide / dose. In another embodiment, the dose is 1000 µg / peptide / dose. In another embodiment, the dose is 1500 µg / peptide / dose. In another embodiment, the dose is 2000 µg / peptide / dose.

In another embodiment, the dose is 10-20 µg / peptide / dose. In another embodiment, the dose is 20-30 µg / peptide / dose.

In another embodiment, the dose is 20-40 µg / peptide / dose. In another embodiment, the dose is 30-60 µg / peptide / dose.

In another embodiment, the dose is 40-80 µg / peptide / dose. In another embodiment, the dose is 50-100 µg / peptide / dose.

In another embodiment, the dose is 50-150 µg / peptide / dose. In another embodiment, the dose is 100-200 µg / peptide / dose.

In another embodiment, the dose is 200-300 µg / peptide / dose. In another embodiment, the dose is 300-400 µg / peptide / dose. In another embodiment, the dose is 400-600 µg / peptide / dose. In another embodiment, the dose is 500

800 µg / peptide / dose. In another embodiment, the dose is 800-1000 µg / peptide / dose. In another embodiment, the dose is

1000-1500 µg / peptide / dose. In another embodiment, the dose is 1500-2000 µg / peptide / dose.

In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.

This invention contemplates various modalities of dose ranges. In another embodiment, the dose is 20 mg per peptide per day. In another embodiment, the dose is 10 mg / peptide / day. In another embodiment, the dose is 30 mg / peptide / day.

In another embodiment, the dose is 40 mg / peptide / day. In another embodiment, the dose is 60 mg / peptide / day. In another embodiment, the dose is 80 mg / peptide / day. In another embodiment, the dose is 100 mg / peptide / day. In another embodiment, the dose is 150 mg / peptide / day. In another embodiment, the dose is 200 mg / peptide / day. In another embodiment, the dose is 300 mg / peptide / day.

In another embodiment, the dose is 400 mg / peptide / day. In another embodiment, the dose is 600 mg / peptide / day. In another embodiment, the dose is 800 mg / peptide / day. In another embodiment, the dose is 1000 mg / peptide / day.

In another embodiment, the dose is 10 mg / peptide / dose. In another embodiment, the dose is 30 mg / peptide / dose. In another embodiment, the dose is 40 mg / peptide / dose. In another embodiment, the dose is 60 mg / peptide / dose. In another embodiment, the dose is 80 mg / peptide / dose. In another embodiment, the dose is 100 mg / peptide / dose. In another embodiment, the dose is 150 mg / peptide / dose. In another embodiment, the dose is 200 mg / peptide / dose. In another embodiment, the dose is 300 mg / peptide / dose. In another embodiment, the dose is 400 mg / peptide / dose. In another embodiment, the dose is 600 mg / peptide / dose. In another embodiment, the dose is 800 mg / peptide / dose. In another embodiment, the dose is 1000 mg / peptide / dose.

In another embodiment, the dose is 10-20 mg / peptide / dose. In another embodiment, the dose is 20-30 mg / peptide / dose. In another embodiment, the dose is 20-40 mg / peptide / dose. In another embodiment, the dose is 30-60 mg / peptide / dose. In another embodiment, the dose is 40-80 mg / peptide / dose. In another embodiment, the dose is 50-100 mg / peptide / dose. In another embodiment, the dose is 50-150 mg / peptide / dose. In another embodiment, the dose is 100-200 mg / peptide / dose. In another embodiment, the dose is 200-300 mg / peptide / dose. In another embodiment, the dose is 300-400 mg / peptide / dose. In another embodiment, the dose is 400-600 mg / peptide / dose. In another embodiment, the dose is 500-800 mg / peptide / dose. In another embodiment, the dose is 800-1000 mg / peptide / dose.

In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.

In another embodiment, the present application describes a kit comprising a peptide, composition or vaccine of the present invention. In another embodiment, the kit further comprises a label or package insert. In another embodiment, the kit is used to detect a WT1-specific CD4 response using a delayed-type hypersensitivity test. In another embodiment, the kit is used for any other method listed in the present description. In another embodiment, the kit is used for any other method known in the art.

Example 1. Materials and Methods

Peptide design. By using three computerized predictive algorithms BIMAS (http: //www-bimas.cit.nih. Gov / cgi-bin / molbio / ken_parker_comboform), SYFPEITHI (http://www.syfpeithi.de/) and RANKPEP (http : //bio.dfci.harvard.edu/Tools/rankpep.html), epitopes were selected for both CD8 and CD4 T cells and started with the native WT1 protein sequences that are capable of inducing an immune response in normal donors. . Heteroclite peptides were designed by altering a single amino acid in the anchor residues of native peptides for class I, resulting in higher predicted binding than their native sequences. Class II peptides were designed by adding flanking residues to class I peptides, to simultaneously stimulate both CD4 and CD8 T cells. Although algorithms can predict many sequences, these models do not predict MHC binding when tested in live cells in 30% of cases (Gomez-Nunez et al., Leuk Res. 2006; 30 (10): 1293-8 ), therefore, in vitro testing is necessary. Furthermore, even if binding is demonstrated, a cytotoxic T cell response may not occur, requiring further in vitro study.

Peptide synthesis. All peptides were purchased and synthesized by Genemed Synthesis, Inc. (San Antonio, TX). The peptides were sterile with a purity of 70% to 90%. The peptides were dissolved in DMSO and diluted in saline to 5 mg / ml and stored at -80 ° C. The control peptides used are: for HLA-DR.B1: DR.B1 binding peptide derived from JAK-2 JAK2-DR (GVCVCGDENILVQEF; SEQ ID NO: 59) or peptide derived from BCR.ABL (IVHSATGFKQSSKALQRPVASDFEP; SEQ ID NO: 60); for HLA-A0201: Ewing's sarcoma derived EW peptide (QLQNPSYDK; SEQ ID NO: 61) and for HLA-A2402: prostate specific membrane antigen (PMSA) derived peptide 624-632 (TYSVSf Ds L; SEQ ID NO: 62).

Cell lines, cytokines and antibodies. Human leukemia cell lines BA25 and HL-60 were used as targets to measure T-cell cytotoxicity. Human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) -1beta, IL- 4, IL-6, IL-15, tumor necrosis factor (TNF) -alpha, and prostaglandin E2 (PGE2) were purchased from R&D Systems (Minneapolis, MN). Beta 2-microglobulin (b2-m) was purchased from Sigma (St. Louis, MO). Antibodies used for immunofluorescence assays, including mAbs for human CD3, CD4, CD8, HLA-A2 (clone BB7.2), and isotype controls were obtained from BD Biosciences (San Diego, CA). Cell isolation kits for CD14 and CD3 were purchased from Miltenyi Biotec. (Bergisch Gladbach, Germany).

T2 assay for peptide binding. T2 cells (TAP, HLA-A0201 +) were incubated overnight at 37 ° C at 1 x 106 cells / ml in FCS-free RPMI medium supplemented with 10 ug / ml human beta-2m (Sigma, St Louis, MO , USA) in the absence (negative control) or presence of peptides at various final concentrations (50, 10 and 2 ug / ml). Brefeldin A (Sigma) at 5 ug / ml was added to the cultures during the last two hours of incubation. T2 cells were then washed and stained with FITC-conjugated anti-HLA-A2.1 (BB7.2) mAb for 30 min at 4 ° C and followed by washing with staining buffer (PBS plus 1% FBS and 0.02% azide). Cell surface HLA-A2 expression was measured by flow cytometry on a FACScalibur (Becton Dickinson) and analyzed with FlowJo 9.6.3 software.

In vitro stimulation and human T cell cultures. Peripheral blood mononuclear cells (PBMC) were obtained from healthy HLA-type donors by Ficoll density centrifugation. CD14 + monocytes were isolated by positive selection using mAb for human CD14 coupled with magnetic beads (Miltenyi Biotec) and used for the first stimulation of T cells. The CD14 fraction of PBMC was used for the isolation of CD3, by separation negative immunomagnetic cell by using a pan-T cell isolation kit (Miltenyi Biotec). The purity of the cells was always greater than 98%. T cells were stimulated for 7 days in the presence of RPMI 1640 supplemented with 5% autologous plasma (AP), 20 ug / ml synthetic peptides, 1 ug / ml B2-m and 10 ng / ml IL-15. Monocyte derived dendritic cells (DC) were generated from CD14 + cells by culturing the cells in RPMI 1640 medium supplemented with 1% AP, 500 units / ml recombinant IL-4 and 1000 units / ml GM-CSF. On days 2 and 4 of incubation, fresh medium with IL-4 and GM-CSF was added or half of the culture medium was replaced. On day 5, 20 ug / ml of the class II peptide was added to the immature DC, for processing. On day 6, a ripening cytokine cocktail was added (Dao et al., Plos One 2009; 4 (8): e6730). On day 7 or 8, T cells were re-stimulated with mature DC, IL-15. In most cases, T cells were stimulated 3 times in the same way, using DC or CD14 + cells as antigen presenting cells (APC). One week after the final challenge, the peptide-specific T-cell response was examined by the enzyme-linked immunosorbent assay (ELISPOT) of IFN-g and cytotoxicity was assessed by the 51-chromium release assay ( Cr).

IFN-g ELISPOT. HA-Multiscreen plates (Millipore) were coated with 100 ul of mouse anti-human IFN-g antibody (10 Ag / ml; clone 1-D1K; Mabtech) in PBS, incubated overnight at 4 ° C, they were washed with PBS to remove unbound antibody and blocked with RPMI 1640/10% autologous plasma (AP) for 2 h at 37 ° C. Purified CD3 + T cells (> 98% purity) were plated with either autologous CD14 + (E: APC ratio 10: 1) or autologous DC (E: APC ratio 30: 1). Various test peptides were added to the wells at 20 ug / ml. Negative control wells contained APC and T cells with no or irrelevant peptides. Positive control wells contained T cells plus APC plus 20 ug / ml phytohaemagglutinin (PHA, Sigma). All conditions were carried out in triplicate. The microtiter plates were incubated for 20 h at 37 ° C and then washed thoroughly with PBS / 0.05% Tween and 100 ul / well of biotinylated detection antibody against human IFN-g (2 ug / ml; clone 7-B6-1; Mabtech). Plates were incubated for an additional 2 hours at 37 ° C and spot development was performed as described (Dao et al., Op. Cit.). The dot counts were determined automatically using a computer-aided video image analyzer with KS ELISPOT 4.0 software (Carl Zeiss Vision).

Chromium release assay. The presence of specific CTLs was measured in a standard chromium release assay as described (Dao et al., Op. Cit.). Briefly, target cells alone or pulsed with 50 ug / ml synthetic peptides for 2 hours (in some cases overnight) at 37 ° C, are labeled with 50 uCi / million cells of Na251CrO4 (NEN Life Science Products , Inc.). After thorough washing, the target cells are incubated with T cells at E: T ratios ranging from 100: 1 to 10: 1. All conditions were carried out in triplicate. The plates were incubated for 4-5 hours at 37 ° C in 5% CO2. Supernatant fluids were harvested and radioactivity was measured in a gamma counter. The percentage of specific lysis was determined from the following formula: [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x 100%. Maximum release was determined by lysis of radiolabeled targets in 1% SDS.

Example 2. Binding of native peptides and their analogues to HLA-A0201 and HLA-A2402

By using a pool of 15 mer overlapping peptides comprising the human WT1 protein to sensitize human T cells in vitro, the sequence 239-248 (NQMNLGATL; SEQ ID No: 5; abbreviated NQM in the present disclosure) has been recently identified as an immunogenic CD8 T cell epitope in the context of HLA-A2402 (Doubrovina et al., Blood 2012; 123 (8): 1633-46). To generate analog peptides with higher immunogenicity, prediction scores of native peptide and possible analogs with various amino acid substitutions at positions 2 and 9 (class I anchor residues) were screened using three available databases. online (BIMAS, RANKPEP and SYFPEITHI). The binding scores predicted from the three databases showed better binding of the native NQMNLGATL peptide (SEQ ID NO: 5) to the HLA-A0201 molecule than to the HLA-A2402 molecule (Table I). When glutamine at position 2 was substituted for leucine, the HLA-A2402 binding score remained at a similar level in all 3 prediction programs. However, a significantly higher binding score was predicted for HLA-A0201. On the other hand, when glutamine at position 2 was substituted for tyrosine, the HLA-A2402 binding score improved dramatically, showing an approximately 90-fold increase in binding according to the BIMAS prediction. All three peptides were predicted to cleave at the c-terminus by the RANKPEP algorithm, suggesting peptide fragment processing. The binding score was checked by substitution with various amino acids at position 9, but none of them showed significantly improved binding compared to substitution at position 2. Therefore, the two analog peptides NLMNLGATL (SEQ ID NO: 6 ; abbreviated NLM or A24-het-1 in the present description) and NYMNLGATL (SEQ ID NO: 7; abbreviated NYM or A24-het-2 in the present description) were selected for further studies.

Table 1. Predictive Binding Scores of HLA-A0201 and A2402 Peptides

Example 3. Binding of peptides to HLA-A0201 and HLA-A2402 molecules

The immunogenicity of MHC class I restricted peptides requires the ability to bind and stabilize MHC class I molecules on the surface of the living cell. Also, computer prediction is only up to 70% accurate; therefore, direct measurement of the strength of the interaction between peptides and HLA-A0201 molecules was sought by using a standard binding and stabilization assay using antigen transport-deficient HLA-A0201 human T2 cells ( negative for TAP2). T2 cells lack TAP function and consequently fail to adequately load class I molecules with antigenic peptides generated in the cytosol. The association of exogenously added peptides with empty and thermolabile HLA-A0201 molecules stabilizes them and results in an increase in the level of surface HLA-A0201 recognizable by a specific anti-HLA-A0201 mAb such as BB7.2.

The T2 binding assay showed that the native NQMNLGATL peptide (SEQ ID NO: 5) did not increase HLA-A2 expression in T2 cells (Figure 1, top panel). However, the peptide analog NLMNLGATL (SEQ ID NO: 6) stabilized the HLA-A2 molecule by showing a dose-dependent increase in HLA-A2 expression, compared to T2 cells without peptide pulsation (Figure 1, center panel). Similar to the native peptide NQMNLGATL, the peptide NYMNLGATL (SEQ ID No. 7) did not increase the expression of HLA-A2 (Figure 1, lower panel). These data confirmed the HLA-A2 binding scores, predicted by the computer-based algorithm.

Example 4. Induction of a peptide-specific CD8 T cell response in the context of HLA-A0201 and A2402 molecules

Although affinity for MHC molecules is necessary for peptide presentation, recognition by T cells of the peptide presented by HLA molecules is another important requirement for eliciting the peptide-specific response. Therefore, using an in vitro stimulation protocol, the new synthetic WT1 peptide analogs were evaluated for their ability to stimulate the peptide-specific T cell response in both HLA-A0201 and A2402 donors.

To expand the peptide-specific T-cell precursors, three to five stimulations were performed in vitro and the response of specific T-cells was measured by IFN-g production, when challenged with individual peptides. The NLMNLGATL peptide induced strong IFN-g secretion that cross-reacted with the native NQMNLGATL peptide. Five T cell stimulations improved the response by showing more IFN-g spots (Figure 2B) than 3 stimulations (Figure 2A). T cells after stimulation with the NLMNLGATL peptide were also tested for cytotoxicity using a 51 Cr release assay. No kill was observed against HL-60 cells that were positive for WT1, but negative for HLA-A2. However, the T cells killed the AML WT1 + and HLA-A0201 + SET-2 cell line and primary leukemia blasts derived from a patient who is HLA-A0201 positive (Figure 3). It was determined whether both the heteroclite peptides NLMNLGATL and NYMNLGATL could induce a better CD8 T cell response in HLA-A2402 donors. The NLMNLGATL peptide was able to induce T cell responses against the native NLMNLGATL and NQMNLGATL peptides, but there was no significant improvement compared to the native NQMNLGATL peptide induced T cell response. In contrast, the NYMNLGATL peptide induced a strong T cell response against itself and the native peptide after 3 stimulations (Figure 4A), but the response was extinguished after 5 rounds of stimulation (Figure 4B), which also showed a weak cross-reactivity with native sequence. These data demonstrated that the heteroclite peptide NLMNLGATL is a strong epitope for CD8 T cells in the context of the HLA-A0201 molecule. The NYMNLGATL peptide, on the other hand, induced a CD8 T cell response in HLA-A0201 positive donors, but the response was not significantly better than that of the NQMNLGATL peptide.

Example 5. Induction of T cell response by HLA-DR.B1 peptides that recognize the CD8 T cell epitope NQMNLGATL

A peptide that combines both the CD4 and CD8 epitopes has been shown to be more effective than the single class I epitope in eliciting an effective immune response for vaccine design, because CD4 T cells can assist CD8 CTLs by fully activating DCs through CD40 / CD40L signaling, as well as by producing IL-2 and IFN-g. Furthermore, if longer peptide stimulated T cells, in which CD8 T cell epitopes are embedded, could recognize the short peptides, it would confirm the processing of CD8 T cell epitopes. Therefore, four HLA-DR.B1 binding peptides were designed that span the epitopes NQMNLGATL and NLMNLGATL, respectively:

DR-Nativo-1: cmtwNQMNLGATLkg (SEQ ID NO: 8)

DR-Nativo-2: wNQMNLGATLkgvaa (SEQ ID NO: 9)

DR-het-1: cmtwNLMNLGATLkg (SEQ ID NO: 14)

DR-het-2: wNLMNLGATLkgvaa (SEQ ID NO: 17)

Since there is no definitive method to predict class II peptide cleavage, two different versions of class II peptides were designed using the BIMAS, SYFPEITHI, and RANKPEP algorithms (Table 2).

When T cells were stimulated with two "heterodite" DR.B1 peptides spanning the NLMNLGATL epitope, they induced T cell responses that were specific for both short and long peptides, shown by IFN-g secretion. Since CD4 peptides induce a more potent response due to their massive cytokine production, the background is usually higher than the CD8 T cell peptide stimulation. Therefore, although both DR-heteroclite peptides induced specific responses, the DR-het-2 peptide showed a clearer response than the DR-het-1 peptide in a donor shown in Figure 5A. It was evident that the responses induced by the DR-het-2 peptide were specific both for the short peptides NQMNGATL and NLMNGATL, as well as for the DR-native 2 and het-2 peptides. More importantly, responses were directed against the irradiated tumor cell line BA-25 (WT1 + A2 +), but not for HL-60 cells that were WT1 + but negative for A0201. Similarly, when T cells were stimulated with short peptides (NQMNLGATL or NLMNGATL) or long peptides as indicated in Figure 5B, only BA-25 cells but not HL-60 cells were killed.

Example 6. Other HLA-DR.B1-binding peptides that recognize the CD8 T-cell epitope NQMNLGATL

In addition to the DR peptides described above, additional HLA-DR.B1 binding peptides spanning the NQMNLGATL, NLMNLGATL, and NLMNLGATL epitopes were designed and evaluated (Table 3):

Table 3. Predictive binding scores of peptides to HLA-DR.B1

(continuation)

Example 7. Generation of peptides derived from the WT1 oncoprotein that bind to human molecules HLA-B7 class I and HLA-Dr class II

Peptides that bind to HLA-B0702 were also designed (Table 4). The following peptide sequences were designed: RQRPHPGAL (B7-Native 1; SEQ ID NO: 34), RLRPHPGAL (B7-het-1; SEQ ID NO: 37), RIRPHPGAL (B7-het-2; SEQ ID NO: 38 ), GALRNPTAC (Native 2; SEQ ID NO: 29), and GALRNPTAL (B7-het-3; SEQ ID NO: 31). The predictive binding scores of these and other variants are shown in Table 4. These peptides were tested in vitro and stimulate heteroclite T cell responses (Figure 6). CD3 T cells from an HLA-B0702 positive donor were stimulated with 2 sets of peptides (five in total) for 5 times in vitro. The specific response for the peptide was measured by the IFN-gamma ELISPOT assay, against a single peptide.

For the first set of peptides, both heteroclite-1 and 2 induced the peptide-specific responses, but the cross-reactivity with native peptide 1 (N1) was stronger for the het-2 peptide than for the het-1 peptide. For the second set of peptides, the heteroclite peptide induced strong IFN-g production, when challenged with the stimulating peptide, but no cross-reactivity was found with the native sequence.

Table 4. Predictive binding scores of peptides B7 to HLA-B7 and other haplotypes.

(continuation)

Based on the finding that the native peptides RQRPHPGAL (p-125 to -117; SEQ ID NO: 34) and GALRNPTAC (p-118 to-110; SEQ ID NO: 29) induce T cell responses in the context of the HLA-B7 molecule, by using HLA binding prediction algorithms, a heteroclite peptide was designed for the GALRNPTAC peptide (SEQ ID NO: 31) and two heteroclite peptides for RQRPHPGAL (s Eq ID NOS: 37 and 38). Based on prediction of binding, these peptides can also stimulate T cells in the context of other HLA haplotypes, such as: A0201, A0301, B8, B1501, B37 and B5101 (Table 4).

Example 8. Generation of peptides derived from the WT1 oncoprotein that bind to human molecules HLA-B35, A0101, A0301, A1101 class I and HLA-DR class II

The peptide QFPNHSFKHEDPMGQ (p170-182) (SEQ ID NO: 39) induces the response of T cells in the context of HLA-DR.B1 0301 and 0401. The short sequences embedded within the long peptide, HSFKHEDPM, induce the response of cells T in the context of B3501. Based on the predictions of the HLA-binding prediction algorithms, a long heteroclite peptide was designed, which is the extension of the short peptide het-B35-1.

The peptide sequences are: class II peptide: DR.B1-03 / 04-Native: QFPNHSFKHEDPM (SEQ ID NO: 42), DR.B1-03 / 04-Het: QFPNHSFKHEDPY (SEQ ID NO: 43; peptides class I: 1. Native: HSFKHEDPM (SEQ ID NO: 40), 2. Het-01 / 03-1: HSFKHEDPY (for A0101 and A0301) (SEQ ID NO: 41) and 3. Het-03 / 11- 1 HSFKHEDPK (for A0301 and A1101) (SEQ ID NO: 42) Heteroclite peptides for the HLA-B3501 haplotype were tested in silico (Table 5).

Example 9. Generation of peptides derived from the WT1 oncoprotein that bind to human molecules HLA-A1, A3, A11 class I and HLA-DR.B1-0401 class II

The peptide KRPFMCAYPGCNK (320-332) (SEQ ID NO: 44) was shown to induce the T cell response in the context of HLA-DR.B1 0401. The short sequence embedded within the long peptide, FMCAYPGCN (SEQ ID NO: 45), induces the T cell response in the context of B35, B7 and A0101 (Table 6). Binding scores of peptides to multiple HLA haplotypes were investigated using prediction algorithms. A long heteroclite peptide was designed, which is the extension of the short peptide het-1. Two short heteroclite peptides were designed that bind better to HLA-A0101, 0301 and 1101. The peptide sequences are: class II peptide: DR.B1-04 Native: KRPFMCAYPGCNK (SEQ ID NO: 44), DR.B1 -04 het: KRPFMCAYPGCYK (SEQ ID NO: 46); class I peptides: 1. Native: FMCAYPGCN (SEQ ID NO: 45), 2. DR.B1-04-Het-1 short: FMCAYPGCY (for A0101) (SEQ ID NO: 47), 3. DR.B1- 04-Het-2-short: FMCAYPGCK (for A0301 and A1101) (SEQ ID NO: 48). KRPFMCAYPGCYK (SEQ ID NO: 46) is the extension of DR.B1-04-het 1 short, FMCAYPGCN (s EQ ID NO: 45), in which the end of the CN sequences is converted to CY.

Table 6. Predictive binding scores of peptides to HLA-DR.B1-0401 and B35, B7, A0101, A0301 and A1101.

Claims (20)

1. An isolated peptide consisting of the amino acid sequence NLMNLGATL (SEQ ID NO: 6). 2. An isolated peptide consisting of the amino acid sequence selected from CMTWNLMNLGATLKG (SEQ ID NO: 14), MTWNLMNLGATLKGV (SEQ ID NO: 15), TWNLMNLGATLKGVA (SEQ ID NO: 16), WNLMNLGATLKGVAA (SEQ ID NO: 17) CMTWNLMNLGATLKGVA (SEQ ID NO: 22). The isolated peptide according to claim 1 or 2, wherein said isolated peptide binds to an HLA class I molecule, an HLA class II molecule or the combination thereof. 4. An isolated class I binding peptide comprising the sequence NLMNLGATL (SEQ ID NO: 6). 5. An isolated class II binding peptide comprising a sequence selected from CMTWNLMNLGATLKG (SEQ ID NO: 14), MTWNLMNLGATLKGV (SEQ ID NO: 15), TWNLMNLGATLKGVA (SEQ ID NO: 16) and WNLMNLGATLKGVAA: 17 (SEQ ID NO: 17 ). 6. A nucleic acid molecule encoding the peptide according to any of claims 1-5. 7. A vector, such as an attenuated virus vector or a Salmonella typhi vector, comprising the nucleic acid molecule according to claim 6. The vector according to claim 7, further encoding any of interleukins 1 to 15, interferon alpha, beta or gamma, tumor necrosis factor, granulocyte macrophage colony stimulating factor (GM-CSF), colony stimulating factor macrophage (M-CSF), granulocyte colony stimulating factor (G-CSF), neutrophil activating protein (NAP), macrophage activating and chemotactic factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia or MIP- Ib, a component of the complement or combinations thereof. 9. A pharmaceutical composition comprising a peptide according to any of claims 1-5, or the nucleic acid molecule according to claim 6 or the vector according to any of claims 7-8, and a carrier, pharmaceutically acceptable carrier or excipient. 10. A vaccine comprising one or more isolated peptides according to claims 1-5. The vaccine according to claim 10 further comprising an antigen presenting cell and / or an adjuvant, a diluent or a carrier. 12. The vaccine according to claim 10 or 11 further comprising at least one additional WT1-derived peptide. The vaccine according to claim 12, wherein said adjuvant is QS21, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, alum, a growth factor, a cytokine, a chemokine, an interleukin, Montanide ISA 51 or GM-CSF. The pharmaceutical composition according to claim 9 or the vaccine according to any of claims 10-13 for use in treating a subject with a WT1-expressing cancer or to reduce an incidence of a WT1-expressing cancer , or their relapse. The pharmaceutical composition or vaccine for use according to claim 14, wherein said WT1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer. , a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a kidney cancer, a Kaposi sarcoma, a sarcoma, a hepatocellular carcinoma, a cancer of Wilms, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS) or a non-small cell lung cancer (NSCLC). 16. The pharmaceutical composition or vaccine for use according to claim 15, wherein said WT1-expressing cancer is mesothelioma. 17. A composition comprising (a) an antigen presenting cell and (b) a peptide according to any of claims 1-5 or the nucleic acid molecule according to claim 6 or the vector according to any of claims 7-8. 18. The use of the peptide according to any of claims 1-5 or the nucleic acid molecule according to claim 6 or the vector according to any of claims 7-8 or the vaccine according to any of the claims 10-13 or the composition according to claim 17 to induce in vitro the formation and proliferation of CTL specific for cancer cells expressing WT1. The use according to claim 18, wherein said WT1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a kidney cancer, a Kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), or non-small cell lung cancer (NSCLC). 20. The use according to claim 18 or 19 wherein the cancer is mesothelioma.